James Kuang-Jan Liao

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• Ali, B., Shenkar, R., Lee, J., Alcazar-Felix, R. J., Thompson, R. E., Stadnik, A., Sader, G., Polster, S. P., Flemming, K. D., Liao, J. K., Sorrentino, M., Girard, R., Hanley, D. F., & Awad, I. A. (2026). Recurrent Symptomatic Hemorrhage in Cerebral Cavernous Malformations After Discontinuation of Atorvastatin or Placebo. Journal of the American Heart Association, 15(Issue 3). doi:10.1161/jaha.125.046943
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  BACKGROUND: A recent randomized prospective controlled trial demonstrated that atorvastatin for up to 2years was safe but did not significantly alter rebleeding in cerebral cavernous malformations. However, any consequences of discontinuing atorvastatin remain unknown. We hypothesized that symptomatic hemorrhage (SH) recurs more frequently in cerebral cavernous malformations after discontinuation of atorvastatin than placebo. METHODS: We conducted a 12-month posttreatment follow-up of patients enrolled in the Atorvastatin Therapy in Cavernous Angiomas with Symptomatic Hemorrhage Exploratory Proof of Concept (AT CASH EPOC) trial (41 randomized to atorvastatin, 39 to placebo) to identify potential recurrent SH after trial drug discontinuation. Every SH was adjudicated by review of imaging and corresponding symptoms. Patients were excluded from follow-up for
• Tai, H. C., Li, X., Park, C. J., Alshaikh, A., Yu, B., Chen, D., Chen, P., & Liao, J. K. (2026). Induction of adipocyte thermogenic program by Rho-associated coiled-coil containing kinase 2. iScience, 29(Issue 4). doi:10.1016/j.isci.2026.115225
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  Beige adipocytes develop within subcutaneous white adipose tissue (sWAT) and contribute to thermogenesis, vital for survival during cold exposure. Rho-associated coiled-coil containing kinase (ROCK) inhibitors have been found to promote white adipocyte differentiation. However, the adipocyte-specific role of ROCK2, the predominant ROCK isoform in adipocytes, in cold-induced thermogenesis is uncertain. Here, we show that ROCK2 in adipocytes is necessary for cold-induced adaptive beiging and thermogenesis. Cold exposure triggered a rapid ROCK activation in both sWAT and interscapular brown adipose tissue (iBAT) from wild-type mice. Mice lacking ROCK2 expression in adipocytes exhibited less cold-induced beige adipocyte development in sWAT and reduced thermogenesis in iBAT. In sWAT, cold-induced upregulation and nuclear translocation of forkhead box protein O1 (FOXO1) were critical for the ROCK2-mediated expression of thermogenic gene uncoupling protein 1 (Ucp1). Our findings indicate that ROCK2 promotes cold-induced beige adipocyte development and thermogenesis, suggesting that ROCK2 is a critical mediator of energy homeostasis.
• Awad, I. A., Alcazar-Felix, R. J., Stadnik, A., Kinkade, S., Jhaveri, A., Lee, J., Hage, S., Iqbal, J., Polster, S. P., Shenkar, R., Treine, K., McBee, N., Ostapkovich, N., Lane, K., Liao, J. K., Sorrentino, M., Lee, C., Flemming, K. D., Girard, R., , Carroll, T. J., et al. (2025). Safety and efficacy of atorvastatin for rebleeding in cerebral cavernous malformations (AT CASH EPOC): a phase 1/2a, randomised placebo-controlled trial. The Lancet Neurology, 24(Issue 4). doi:10.1016/s1474-4422(25)00036-5
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  Background: Cerebral cavernous malformations (CCMs) carry a high risk of rebleeding after symptomatic haemorrhage, with serious clinical sequelae. Atorvastatin was shown to prevent CCM growth and bleeding in animal models. We aimed to assess the safety and efficacy of atorvastatin on rebleeding in patients with CCMs after a symptomatic haemorrhage. Methods: We did a phase 1/2a randomised trial at the University of Chicago's CCM Center of Excellence. Patients aged 18–80 years with untreated CCMs who had had symptomatic bleeding from a CCM lesion within the previous year were eligible. Patients were randomly allocated (1:1) to oral atorvastatin (80 mg daily for 2 years) or matching placebo. Investigators, clinical staff, and participants were masked to the assigned treatment. The primary efficacy outcome was the percentage change in mean lesional iron deposition per year, measured by quantitative susceptibility mapping (QSM) on MRI and averaged over 2 years; a decrease would signal potential benefit and an increase a safety concern. The primary efficacy outcome was analysed in the modified intention-to-treat cohort, including patients with at least one annual paired QSM assessment. Safety outcomes included rates of bleeds and serious adverse events necessitating drug discontinuation. This trial is registered at ClinicalTrials.gov (NCT02603328) and is completed. Findings: Between July 25, 2018, and July 22, 2022, 326 patients were assessed for eligibility, and 80 patients were allocated either atorvastatin (n=41) or placebo (n=39). 29 (36%) patients were male and 51 (64%) were female. 64 (80%) patients (33 in the atorvastatin group and 31 in the placebo group) had at least one annual paired QSM assessment and were included in the modified intention-to-treat analyses. The mean annual percentage change in lesional QSM was 10·88 (SE 7·29) with atorvastatin versus 12·09 (SE 7·54) with placebo (treatment effect –1·22, 95% CI –22·25 to 19·81; p=0·91). Symptomatic haemorrhage was reported in six patients assigned atorvastatin and seven patients assigned placebo (relative risk 0·81, 95% CI 0·31 to 2·13). No patients had a serious adverse event requiring drug discontinuation and no deaths were recorded. Interpretation: For people with symptomatic haemorrhage caused by CCMs, atorvastatin did not affect the mean change in lesional iron deposition on brain MRI over 2 years when compared with placebo. Atorvastatin was well tolerated and no safety concerns were noted. The study provides a useful framework for biomarker driven drug assessment in a rare disease. Funding: US National Institutes of Health.
• Lange, M., Francis, C., Furtado, J., Granger, J., Kim, Y. B., Liao, J. K., Akar, F. G., & Eichmann, A. (2025). Endothelial Rho kinase controls blood vessel integrity and angiogenesis. Cardiovascular Research, 121(Issue 18). doi:10.1093/cvr/cvaf246
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  Aims The Rho kinases 1 and 2 (ROCK1/2) are serine-threonine specific protein kinases that control actin cytoskeleton dynamics. They are expressed in all cells throughout the body, including cardiomyocytes, smooth muscle cells and endothelial cells (ECs), and are intimately involved in cardiovascular health and disease. Pharmacological ROCK inhibition is beneficial in mouse models of hypertension, atherosclerosis, and neointimal thickening that display overactivated ROCK. However, the consequences of endothelial ROCK signalling deficiency in vivo remained unknown. Methods and results To address this issue, we generated Cdh5CreERT2 driven, tamoxifen inducible loss of function alleles of ROCK1 and ROCK2 and analysed mouse survival and vascular defects through cellular, biochemical, and molecular biology approaches. We observed that postnatal or adult loss of endothelial ROCK1 and 2 was lethal within a week. Mice developed multi-organ haemorrhage along with loss of vascular integrity. ECs displayed deficient cytoskeletal actin polymerization that prevented focal adhesion formation and disrupted junctional integrity. Retinal sprouting angiogenesis was also perturbed, as sprouting vessels exhibited lack of polymerized actin and defective lumen formation. In a three-dimensional endothelial sprouting assay, combined knockdown of ROCK1/2 or individual knockdown of ROCK2 but not ROCK1 led to reduced sprouting, lumenization and cell polarization defects caused by defective actin and altered VE-cadherin dynamics. The isoform specific role of endothelial ROCK2 correlated with ROCK2 substrate specificity for FAK and LIMK. By analysing single and three allele mutants we show that one intact allele of ROCK2 is sufficient to maintain vascular integrity in vivo. Conclusion Endothelial ROCK1 and 2 maintain junctional integrity and ensure proper angiogenesis and lumen formation. The presence of one allele of ROCK2 is sufficient to maintain vascular growth and integrity. These data indicate the need for careful consideration when using ROCK inhibitors in disease settings.
• Ullah, K., Ai, L., Li, Y., Liu, L., Zhang, Q., Pan, K., Humayun, Z., Piao, L., Sitikov, A., Zhao, Q., Su, Q., Sharp, W., Fang, Y., Wu, D., Liao, J. K., & Wu, R. (2025). ARNT-dependent HIF-2α signaling protects cardiac microvascular barrier integrity and heart function post-myocardial infarction. Communications Biology, 8(Issue 1). doi:10.1038/s42003-025-07753-1
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  Myocardial infarction (MI) compromises the cardiac microvascular endothelial barrier, increasing leakage and inflammation. HIF2α, predominantly expressed in cardiac endothelial cells during ischemia, has an unclear role in barrier function during MI. Here, we show that inducible, adult endothelial-specific deletion of Hif2α in mice leads to increased mortality, cardiac leakage, inflammation, reduced heart function, and adverse remodeling after MI. In parallel, human cardiac microvascular endothelial cells (HCMVECs) lacking HIF2α display impaired barrier integrity, reduced tight-junction proteins, increased cell death, and elevated IL-6 levels, effects that are alleviated by overexpressing ARNT, a key partner of HIF2α under hypoxic conditions. Interestingly, ARNT, but not HIF2α, directly binds the IL-6 promoter to suppress its expression. These findings suggest the HIF2α/ARNT axis as a protective mechanism in heart failure post-MI and identify potential therapeutic targets to support cardiac function. (Figure presented.)
• Fukuma, N., Tokiwa, H., Numata, G., Ueda, K., Liu, P. Y., Tajima, M., Otsu, Y., Kariya, T., Hiroi, Y., Liao, J. K., Komuro, I., & Takimoto, E. (2024). Endothelial oestrogen–myocardial cyclic guanosine monophosphate axis critically determines angiogenesis and cardiac performance during pressure overload. Cardiovascular Research, 120(Issue 15). doi:10.1093/cvr/cvae202
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  Aims. Oestrogen exerts beneficial cardiovascular effects by binding to specific receptors on various cells to activate nuclear and non-nuclear actions. Oestrogen receptor α (ERα) non-nuclear signalling confers protection against heart failure remodelling, involving myocardial cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase G (PKG) activation; however, its tissue-specific role remains elusive. Herein, we examine the cell type–specific role of ERα non-nuclear signalling in oestrogen-conferred protection against heart failure. Methods and results. We first assessed the tissue-specific impacts of ERα on the cardiac benefits derived from oestrogen, utilizing endothelial ERα deletion (ERαf/f/Tie2Cre+) and myocyte ERα deletion (ERαf/f/αMHCCre+) female mice. Female mice were ovariectomized and the effect of estradiol (E2) was assessed in hearts exposed to 3 weeks of pressure overload [transverse aortic constriction (TAC)]. E2 failed to improve cardiac function in ERαf/f/Tie2Cre+ TAC hearts but provided benefits in ERαf/f/αMHCCre+ TAC hearts, indicating that endothelial ERα is essential. We next assessed the role of non-nuclear signalling in endothelial cells (ECs), employing animals with endothelial-specific inactivation of ERα non-nuclear signalling (ERαKI/KI/Tie2Cre+). Female ovariectomized mice were supplemented with E2 and subjected to 3-week TAC. ERαKI/KI/Tie2Cre+TAC hearts revealed exacerbated cardiac dysfunction and reduced myocardial PKG activity as compared to littermate TAC hearts, which were associated with attenuated myocardial induction of vascular endothelial growth factor (VEGF) and angiogenesis as assessed by CD31-stained capillary density. This phenotype of ERαKI/KI/Tie2Cre+was rescued by myocardial PKG activation from chronic treatment with a soluble guanylate cyclase (sGC) stimulator. We performed co-culture experiments to determine endothelial–cardiomyocyte interactions. VEGF induction by E2 in cardiac myocytes required a co-existence of intact endothelial ERα signalling in a nitric oxide synthase-dependent manner. On the other hand, VEGF was induced in myocytes directly with an sGC stimulator in the absence of ECs. Conclusion. An endothelial oestrogen–myocardial cGMP axis stimulates angiogenic response and improves cardiac performance during pressure overload.
• Maas-Bauer, K., Stell, A. V., Yan, K. L., de Vega, E., Vinnakota, J. M., Unger, S., Núñez, N., Norona, J., Talvard-Balland, N., Koßmann, S., Schwan, C., Miething, C., Martens, U. S., Shoumariyeh, K., Nestor, R. P., Duquesne, S., Hanke, K., Rackiewicz, M., Hu, Z., , El Khawanky, N., et al. (2024). ROCK1/2 signaling contributes to corticosteroid-refractory acute graft-versus-host disease. Nature Communications, 15(Issue 1). doi:10.1038/s41467-024-44703-7
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  Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1β, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings.
• Liu, P. Y., Fukuma, N., Hiroi, Y., Kunita, A., Tokiwa, H., Ueda, K., Kariya, T., Numata, G., Adachi, Y., Tajima, M., Toyoda, M., Li, Y., Noma, K., Harada, M., Toko, H., Ushiku, T., Kanai, Y., Takimoto, E., Liao, J. K., & Komuro, I. (2023). Tie2-Cre–Induced Inactivation of Non-Nuclear Estrogen Receptor-α Signaling Abrogates Estrogen Protection Against Vascular Injury. JACC: Basic to Translational Science, 8(Issue 1). doi:10.1016/j.jacbts.2022.07.001
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  Using the Cre-loxP system, we generated the first mouse model in which estrogen receptor-α non-nuclear signaling was inactivated in endothelial cells. Estrogen protection against mechanical vascular injury was impaired in this model. This result indicates the pivotal role of endothelial estrogen receptor-α non-nuclear signaling in the vasculoprotective effects of estrogen.
• Zarkada, G., Chen, X., Zhou, X., Lange, M., Zeng, L., Lv, W., Zhang, X., Li, Y., Zhou, W., Liu, K., Chen, D., Ricard, N., Liao, J., Kim, Y., Benedito, R., Claesson-Welsh, L., Alitalo, K., Simons, M., Ju, R., , Li, X., et al. (2023). Chylomicrons Regulate Lacteal Permeability and Intestinal Lipid Absorption. Circulation Research, 133(4), 333-349. doi:10.1161/circresaha.123.322607
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  Background: Lymphatic vessels are responsible for tissue drainage, and their malfunction is associated with chronic diseases. Lymph uptake occurs via specialized open cell-cell junctions between capillary lymphatic endothelial cells (LECs), whereas closed junctions in collecting LECs prevent lymph leakage. LEC junctions are known to dynamically remodel in development and disease, but how lymphatic permeability is regulated remains poorly understood. Methods: We used various genetically engineered mouse models in combination with cellular, biochemical, and molecular biology approaches to elucidate the signaling pathways regulating junction morphology and function in lymphatic capillaries. Results: By studying the permeability of intestinal lacteal capillaries to lipoprotein particles known as chylomicrons, we show that ROCK (Rho-associated kinase)-dependent cytoskeletal contractility is a fundamental mechanism of LEC permeability regulation. We show that chylomicron-derived lipids trigger neonatal lacteal junction opening via ROCK-dependent contraction of junction-anchored stress fibers. LEC-specific ROCK deletion abolished junction opening and plasma lipid uptake. Chylomicrons additionally inhibited VEGF (vascular endothelial growth factor)-A signaling. We show that VEGF-A antagonizes LEC junction opening via VEGFR (VEGF receptor) 2 and VEGFR3-dependent PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) activation of the small GTPase RAC1 (Rac family small GTPase 1), thereby restricting RhoA (Ras homolog family member A)/ROCK-mediated cytoskeleton contraction. Conclusions: Our results reveal that antagonistic inputs into ROCK-dependent cytoskeleton contractions regulate the interconversion of lymphatic junctions in the intestine and in other tissues, providing a tunable mechanism to control the lymphatic barrier.
• Li, M., Lyu, X., Liao, J., Werth, V. P., & Liu, M. L. (2022). Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation. Theranostics, 12(Issue 5). doi:10.7150/thno.66457
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  Objective: Ultraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKCα-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKCα nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation. Methods: We studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKCα nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation. Results: Our time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKCα in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKCα nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNFα, IFNγ, and IFNα in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency. Conclusions: ROCK regulated NETosis through modulation of PKCα nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB-induced skin inflammation by attenuation of NETosis and NET-associated cytokines.
• Yu, B., Osman, A. E., Sladojevic, N., Prabhu, N., Tai, H. C., Chen, D., Perla, G., Park, L., Larson, R. A., & Liao, J. K. (2022). Involvement of Rho-Associated Coiled-Coil Containing Kinase (ROCK) in BCR-ABL1 Tyrosine Kinase Inhibitor Cardiovascular Toxicity. JACC: CardioOncology, 4(Issue 3). doi:10.1016/j.jaccao.2022.06.004
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  Background: Second- and third-generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) are associated with cardiovascular adverse events (CVAEs) in patients with Philadelphia chromosome–positive (Ph+) leukemia. Objectives: We hypothesized that second- and third-generation BCR-ABL1 TKIs may cause CVAEs through the activation of Rho-associated coiled-coil containing kinase (ROCK). Methods: Peripheral blood mononuclear cells from 53 Ph+ patients on TKIs and 15 control patients without Ph+ leukemia were assessed for ROCK activity through capillary electrophoresis (median follow-up = 26 months [Q1-Q3: 5-37 months]). We also investigated the effects of TKIs and ROCK on endothelial dysfunction in vitro, which could contribute to CVAEs. Results: Patients receiving second- and third-generation TKIs had 1.6-fold greater ROCK activity compared with patients receiving imatinib and control patients. Elevated ROCK activity was associated with an increased incidence of CVAEs in Ph+ leukemia patients. In endothelial cells in vitro, we found that dasatinib and ponatinib treatment led to changes in actin intensity and endothelial permeability, which can be reversed by pharmacologic inhibition of ROCK. Ponatinib led to decreased cell proliferation, but this was not accompanied by senescence. Dasatinib and ponatinib treatment led to phosphor-inhibition of endothelial nitric oxide synthase and decreased nitric oxide production. ROCK inhibition reversed endothelial permeability and endothelial nitric oxide synthase–related endothelial dysfunction. Imatinib and nilotinib induce phosphorylation of p190RhoGAP. Conclusions: Our findings suggest ROCK activity may be a prognostic indicator of CVAEs in patients receiving BCR-ABL1 TKIs. With further study, ROCK inhibition may be a promising approach to reduce the incidence of CVAEs associated with second- and third-generation BCR-ABL1 TKIs.
• Anyanwu, E. C., Chua, R. F., Besser, S. A., Sun, D., Liao, J. K., & Tabit, C. E. (2021). SALAD-BAAR: A numerical risk score for hospital admission or emergency department presentation in ambulatory patients with cardiovascular disease. Clinical Cardiology, 44(Issue 2). doi:10.1002/clc.23525
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  Background: While many interventions to reduce hospital admissions and emergency department (ED) visits for patients with cardiovascular disease have been developed, identifying ambulatory cardiac patients at high risk for admission can be challenging. Hypothesis: A computational model based on readily accessible clinical data can identify patients at risk for admission. Methods: Electronic health record (EHR) data from a tertiary referral center were used to generate decision tree and logistic regression models. International Classification of Disease (ICD) codes, labs, admissions, medications, vital signs, and socioenvironmental variables were used to model risk for ED presentation or hospital admission within 90 days following a cardiology clinic visit. Model training and testing were performed with a 70:30 data split. The final model was then prospectively validated. Results: A total of 9326 patients and 46 465 clinic visits were analyzed. A decision tree model using 75 patient characteristics achieved an area under the curve (AUC) of 0.75 and a logistic regression model achieved an AUC of 0.73. A simplified 9-feature model based on logistic regression odds ratios achieved an AUC of 0.72. A further simplified numerical score assigning 1 or 2 points to each variable achieved an AUC of 0.66, specificity of 0.75, and sensitivity of 0.58. Prospectively, this final model maintained its predictive performance (AUC 0.63–0.60). Conclusion: Nine patient characteristics from routine EHR data can be used to inform a highly specific model for hospital admission or ED presentation in cardiac patients. This model can be simplified to a risk score that is easily calculated and retains predictive performance.
• Li, Y., Tai, H. C., Sladojevic, N., Kim, H. H., & Liao, J. K. (2021). Vascular stiffening mediated by rho-associated coiled-coil containing kinase isoforms. Journal of the American Heart Association, 10(Issue 20). doi:10.1161/jaha.121.022568
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  BACKGROUND: The pathogenesis of vascular stiffening and hypertension is marked by non-compliance of vessel wall because of deposition of collagen fibers, loss of elastin fibers, and increased vascular thickening. Rho/Rho-associated coiled-coil containing kinases 1 and 2 (ROCK1 and ROCK2) have been shown to regulate cellular contraction and vascular remodeling. However, the role of ROCK isoforms in mediating pathogenesis of vascular stiffening and hypertension is not known. METHODS AND RESULTS: Hemizygous Rock mice (Rock1+/− and Rock2+/−) were used to determine the role of ROCK1 and ROCK2 in age-related vascular dysfunction. Both ROCK activity and aortic stiffness increased to a greater extent with age in wild-type mice compared with that of Rock1+/− and Rock2+/− mice. As a model for age-related vascular stiffening, we administered angiotensin II (500 ng/kg per minute) combined with nitric oxide synthase inhibitor, L-Nω-nitroarginine methyl ester (0.5 g/L) for 4 weeks to 12-week-old male Rock1+/− and Rock2+/− mice. Similar to advancing age, angiotensin II/L-Nω-nitroarginine methyl ester caused increased blood pressure, aortic stiffening, and vascular remodeling, which were attenuated in Rock2+/−, and to a lesser extent, Rock1+/− mice. The reduction of aortic stiffening in Rock2+/− mice was accompanied by decreased collagen deposition, relatively preserved elastin content, and less aortic wall hypertrophy. Indeed, the upregulation of collagen I by transforming growth factor-β1 or angiotensin II was greatly attenuated in Rock2−/− mouse embryonic fibroblasts. CONCLUSIONS: These findings indicate that ROCK1 and ROCK2 mediate both age-related and pharmacologically induced aor-tic stiffening, and suggest that inhibition of ROCK2, and to a lesser extent ROCK1, may have therapeutic benefits in preventing age-related vascular stiffening.
• Wei, J., Liao, J. K., & Bairey Merz, C. N. (2021). Challenging Statin Pleiotropy: Preeclampsia. Circulation, 144(Issue 9). doi:10.1161/circulationaha.121.056140
• Zhang, Q., Zheng, M., Betancourt, C. E., Liu, L., Sitikov, A., Sladojevic, N., Zhao, Q., Zhang, J. H., Liao, J. K., & Wu, R. (2021). Increase in blood-brain barrier (BBB) permeability is regulated by MMP3 via the erk signaling pathway. Oxidative Medicine and Cellular Longevity, 2021(Issue). doi:10.1155/2021/6655122
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  Background. The blood-brain barrier (BBB) regulates the exchange of molecules between the brain and peripheral blood and is composed primarily of microvascular endothelial cells (BMVECs), which form the lining of cerebral blood vessels and are linked via tight junctions (TJs). The BBB is regulated by components of the extracellular matrix (ECM), and matrix metalloproteinase 3 (MMP3) remodels the ECM's basal lamina, which forms part of the BBB. Oxidative stress is implicated in activation of MMPs and impaired BBB. Thus, we investigated whether MMP3 modulates BBB permeability. Methods. Experiments included in vivo assessments of isoflurane anesthesia and dye extravasation from brain in wild-type (WT) and MMP3-deficient (MMP3-KO) mice, as well as in vitro assessments of the integrity of monolayers of WT and MMP3-KO BMVECs and the expression of junction proteins. Results. Compared to WT mice, measurements of isoflurane usage and anesthesia induction time were higher in MMP3-KO mice and lower in WT that had been treated with MMP3 (WT+MMP3), while anesthesia emergence times were shorter in MMP3-KO mice and longer in WT+MMP3 mice than in WT. Extravasation of systemically administered dyes was also lower in MMP3-KO mouse brains and higher in WT+MMP3 mouse brains, than in the brains of WT mice. The results from both TEER and Transwell assays indicated that MMP3 deficiency (or inhibition) increased, while MMP3 upregulation reduced barrier integrity in either BMVEC or the coculture. MMP3 deficiency also increased the abundance of TJs and VEcadherin proteins in BMVECs, and the protein abundance declined when MMP3 activity was upregulated in BMVECs, but not when the cells were treated with an inhibitor of extracellular signal related-kinase (ERK). Conclusion. MMP3 increases BBB permeability following the administration of isoflurane by upregulating the ERK signaling pathway, which subsequently reduces TJ and VE-cadherin proteins in BMVECs.
• McKerracher, L., Shenkar, R., Abbinanti, M., Cao, Y., Peiper, A., Liao, J. K., Lightle, R., Moore, T., Hobson, N., Gallione, C., Ruschel, J., Koskimäki, J., Girard, R., Rosen, K., Marchuk, D. A., & Awad, I. A. (2020). A Brain-Targeted Orally Available ROCK2 Inhibitor Benefits Mild and Aggressive Cavernous Angioma Disease. Translational Stroke Research, 11(Issue 3). doi:10.1007/s12975-019-00725-8
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  Cavernous angioma (CA) is a vascular pathology caused by loss of function in one of the 3 CA genes (CCM1, CCM2, and CCM3) that result in rho kinase (ROCK) activation. We investigated a novel ROCK2 selective inhibitor for the ability to reduce brain lesion formation, growth, and maturation. We used genetic methods to explore the use of a ROCK2-selective kinase inhibitor to reduce growth and hemorrhage of CAs. The role of ROCK2 in CA was investigated by crossing Rock1 or Rock2 hemizygous mice with Ccm1 or Ccm3 hemizygous mice, and we found reduced lesions in the Rock2 hemizygous mice. A ROCK2-selective inhibitor, BA-1049 was used to investigate efficacy in reducing CA lesions after oral administration to Ccm1+/− and Ccm3+/− mice that were bred into a mutator background. After assessing the dose range effective to target brain endothelial cells in an ischemic brain model, Ccm1+/− and Ccm3+/− transgenic mice were treated for 3 (Ccm3+/−) or 4 months (Ccm1+/−), concurrently, randomized to receive one of three doses of BA-1049 in drinking water, or placebo. Lesion volumes were assessed by micro-computed tomography. BA-1049 reduced activation of ROCK2 in Ccm3+/−Trp53−/− lesions. Ccm1+/−Msh2−/− (n=68) and Ccm3+/−Trp53−/− (n=71) mice treated with BA-1049 or placebo showed a significant dose-dependent reduction in lesion volume after treatment with BA-1049, and a reduction in hemorrhage (iron deposition) near lesions at all doses. These translational studies show that BA-1049 is a promising therapeutic agent for the treatment of CA, a disease with no current treatment except surgical removal of the brain lesions.
• Ricker, E., Chinenov, Y., Pannellini, T., Flores-Castro, D., Ye, C., Gupta, S., Manni, M., Liao, J. K., & Pernis, A. B. (2020). Serine-threonine kinase ROCK2 regulates germinal center B cell positioning and cholesterol biosynthesis. Journal of Clinical Investigation, 130(Issue 7). doi:10.1172/jci132414
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  Germinal center (GC) responses require B cells to respond to a dynamic set of intercellular and microenvironmental signals that instruct B cell positioning, differentiation, and metabolic reprogramming. RHO-associated coiled-coil-containing protein kinase 2 (ROCK2), a serine-threonine kinase that can be therapeutically targeted by ROCK inhibitors or statins, is a key downstream effector of RHOA GTPases. Although RHOA-mediated pathways are emerging as critical regulators of GC responses, the role of ROCK2 in B cells is unknown. Here, we found that ROCK2 was activated in response to key T cell signals like CD40 and IL-21 and that it regulated GC formation and maintenance. RNA-Seq analyses revealed that ROCK2 controlled a unique transcriptional program in GC B cells that promoted optimal GC polarization and cholesterol biosynthesis. ROCK2 regulated this program by restraining AKT activation and subsequently enhancing FOXO1 activity. ATAC-Seq (assay for transposase-accessible chromatin with high-throughput sequencing) and biochemical analyses revealed that the effects of ROCK2 on cholesterol biosynthesis were instead mediated via a novel mechanism. ROCK2 directly phosphorylated interferon regulatory factor 8 (IRF8), a crucial mediator of GC responses, and promoted its interaction with sterol regulatory element-binding transcription factor 2 (SREBP2) at key regulatory regions controlling the expression of cholesterol biosynthetic enzymes, resulting in optimal recruitment of SREBP2 at these sites. These findings thus uncover ROCK2 as a multifaceted and therapeutically targetable regulator of GC responses.
• Sladojevic, N., Yu, B., & Liao, J. K. (2020). Regulator of g-protein signaling 5 maintains brain endothelial cell function in focal cerebral ischemia. Journal of the American Heart Association, 9(Issue 18). doi:10.1161/jaha.120.017533
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  BACKGROUND: Regulator of G-protein signaling 5 (RGS5) is a negative modulator of G-protein–coupled receptors. The role of RGS5 in brain endothelial cells is not known. We hypothesized that RGS5 in brain microvascular endothelial cells may be an important mediator of blood-brain barrier function and stroke severity after focal cerebral ischemia. METHODS AND RESULTS: Using a transient middle cerebral artery occlusion model, we found that mice with global and endothelial-specific deletion of Rgs5 exhibited larger cerebral infarct size, greater neurological motor deficits, and increased brain edema. In our in vitro models, we observed increased Gq activity and elevated intracellular Ca2+ levels in brain endothelial cells. Furthermore, the loss of endothelial RGS5 leads to decreased endothelial NO synthase expression and phosphorylation, relocalization of endothelial tight junction proteins, and increased cell permeability. Indeed, RGS5 deficiency leads to increased Rho-associated kinase and myosin light chain kinase activity, which were partially reversed in our in vitro model by pharmacological inhibition of Gq, metabotropic glutamate receptor 1, and ligand-gated ionotropic glutamate receptor. CONCLUSIONS: Our findings indicate that endothelial RGS5 plays a novel neuroprotective role in focal cerebral ischemia. Loss of endothelial RGS5 leads to hyperresponsiveness to glutamate signaling pathways, enhanced Rho-associated kinase– and myosin light chain kinase–mediated actin-cytoskeleton reorganization, endothelial dysfunction, tight junction protein relocalization, increased blood-brain barrier permeability, and greater stroke severity. These findings suggest that preservation of endothelial RGS5 may be an important therapeutic strategy for maintaining blood-brain barrier integrity and limiting the severity of ischemic stroke.
• Vohra, A. S., Chua, R. F., Besser, S. A., Alcain, C. F., Basnet, S., Battle, B., Coplan, M. J., Liao, J. K., & Tabit, C. E. (2020). Community Health Workers Reduce Rehospitalizations and Emergency Department Visits for Low-Socioeconomic Urban Patients with Heart Failure. Critical Pathways in Cardiology, 19(Issue 3). doi:10.1097/hpc.0000000000000220
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  Background: Low-socioeconomic, urban, minority patients with heart failure (HF) often have unique barriers to care. Community health workers (CHWs) are specially trained laypeople who serve as liaisons between underserved communities and the health system. It is not known whether CHWs improve outcomes in low-socioeconomic, urban, minority patients with HF. Hypothesis: CHWs reduce rehospitalizations, emergency department (ED) visits, and healthcare costs for low-socioeconomic urban patients with HF. Methods: Patients admitted with acute decompensated HF were assigned to receive weekly visits by CHW after discharge. Patients were propensity score matched with controls who received usual care. HF-related rehospitalizations, ED visits, and inpatient costs were compared for 12 months following index admission versus the same period before. Results: Twenty-eight patients who received weekly visits from a CHW for 12 months after discharge were matched with 28 control patients who did not receive CHWs. Patients who received a CHW had a 75% decrease in HF-related ED visits (0.71 vs. 0.18 visits per patient, P < 0.001), an 89% decrease in HF-related readmissions (0.64 vs. 0.07 admissions per patient, P < 0.005), and a significant decrease in inpatient cost for HF-related visits. In controls receiving usual care, there was no significant change in hospitalizations, ED visits, or costs. Conclusions: In conclusion, CHWs are associated with reduced rehospitalizations, ED visits, and inpatient costs in low-socioeconomic, urban, minority patients with HF. CHWs may be a cost-effective method to reduce health care utilization and improve outcomes for this population.
• Kishimoto, S., Oki, K., Maruhashi, T., Kajikawa, M., Matsui, S., Hashimoto, H., Takaeko, Y., Kihara, Y., Chayama, K., Goto, C., Aibara, Y., Yusoff, F. M., Nakashima, A., Noma, K., Liao, J. K., & Higashi, Y. (2019). Eplerenone improves endothelial function and arterial stiffness and inhibits Rho-associated kinase activity in patients with idiopathic hyperaldosteronism: A pilot study. Journal of Hypertension, 37(Issue 5). doi:10.1097/hjh.0000000000001989
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  Objective: Primary aldosteronism is one of the most common cause of secondary hypertension. It is well known that the incidence of cardiovascular events is higher in patients with primary aldosteronism than in patients with essential hypertension. In a previous study, we showed that aldosterone-producing adenoma is associated with vascular function and structure. The aim of this study was to evaluate the effects of eplerenone on vascular function in the macrovasculature and microvasculature, arterial stiffness and Rho-associated kinase (ROCK) activity in patients with idiopathic hyperaldosteronism (IHA). Methods: Vascular function, including reactive hyperemia index (RHI), flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID), arterial stiffness including brachial-ankle pulse wave velocity (baPWV) and brachial intima-media thickness (IMT) and ROCK activity in peripheral leukocytes were measured before and after 12 weeks of treatment with eplerenone in 50 patients with IHA. Results: After 12 weeks, eplerenone decreased the aldosterone renin ratio but did not alter SBP and DBP. Eplerenone treatment increased log RHI from 0.56±0.25 to 0.69±0.25 (P
• Polster, S. P., Stadnik, A., Akers, A. L., Cao, Y., Christoforidis, G. A., Fam, M. D., Flemming, K. D., Girard, R., Hobson, N., Koenig, J. I., Koskimäki, J., Lane, K., Liao, J. K., Lee, C., Lyne, S. B., McBee, N., Morrison, L., Piedad, K., Shenkar, R., , Sorrentino, M., et al. (2019). Atorvastatin Treatment of Cavernous Angiomas with Symptomatic Hemorrhage Exploratory Proof of Concept (AT CASH EPOC) Trial. Clinical Neurosurgery, 85(Issue 6). doi:10.1093/neuros/nyy539
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  BACKGROUND: More than a million Americans harbor a cerebral cavernous angioma (CA), and those who suffer a prior symptomatic hemorrhage have an exceptionally high rebleeding risk. Preclinical studies show that atorvastatin blunts CA lesion development and hemorrhage through inhibiting RhoA kinase (ROCK), suggesting it may confer a therapeutic benefit. OBJECTIVE: To evaluate whether atorvastatin produces a difference compared to placebo in lesional iron deposition as assessed by quantitative susceptibility mapping (QSM) on magnetic resonance imaging in CAs that have demonstrated a symptomatic hemorrhage in the prior year. Secondary aims shall assess effects on vascular permeability, ROCK activity in peripheral leukocytes, signal effects on clinical outcomes, adverse events, and prespecified subgroups. METHODS: The phase I/IIa placebo-controlled, double-blinded, single-site clinical trial aims to enroll 80 subjects randomized 1-1 to atorvastatin (starting dose 80 mg PO daily) or placebo. Dosing shall continue for 24-mo or until reaching a safety endpoint. EXPECTED OUTCOMES: The trial is powered to detect an absolute difference of 20% in the mean percent change in lesional QSM per year (2-tailed, power 0.9, alpha 0.05). A decrease in QSM change would be a signal of potential benefit, and an increase would signal a safety concern with the drug. DISCUSSION: With firm mechanistic rationale, rigorous preclinical discoveries, and biomarker validations, the trial shall explore a proof of concept effect of a widely used repurposed drug in stabilizing CAs after a symptomatic hemorrhage. This will be the first clinical trial of a drug aimed at altering rebleeding in CA.
• Shenkar, R., Peiper, A., Pardo, H., Moore, T., Lightle, R., Girard, R., Hobson, N., Polster, S. P., Koskimäki, J., Zhang, D., Lyne, S. B., Cao, Y., Chaudagar, K., Saadat, L., Gallione, C., Pytel, P., Liao, J. K., Marchuk, D., & Awad, I. A. (2019). Rho kinase inhibition blunts lesion development and hemorrhage in murine models of aggressive Pdcd10/Ccm3 disease. Stroke, 50(Issue 3). doi:10.1161/strokeaha.118.024058
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  Background and Purpose - Previously, murine models Krit1Msh2-/- and Ccm2-Trp53-/- showed a reduction or no effect on cerebral cavernous malformation (CCM) burden and favorable effects on lesional hemorrhage by the robust Rock (Rho-associated protein kinase) inhibitor fasudil and by simvastatin (a weak pleiotropic inhibitor of Rock). Herein, we concurrently investigated treatment of the more aggressive Pdcd10/Ccm3 model with fasudil, simvastatin, and higher dose atorvastatin to determined effectiveness of Rock inhibition. Methods - The murine models, Pdcd10-Trp53-/- and Pdcd10-Msh2-/-, were contemporaneously treated from weaning to 5 months of age with fasudil (100 mg/kg per day in drinking water, n=9), simvastatin (40 mg/kg per day in chow, n=11), atorvastatin (80 mg/kg per day in chow, n=10), or with placebo (n=16). We assessed CCM volume in mouse brains by microcomputed tomography. Lesion burden was calculated as lesion volume normalized to total brain volume. We analyzed chronic hemorrhage in CCM lesions by quantitative intensity of Perls staining in brain sections. Results - The Pdcd10-Trp53-/-/Msh2-/- models showed a mean CCM lesion burden per mouse reduction from 0.0091 in placebos to 0.0042 (P=0.027) by fasudil, and to 0.0047 (P=0.025) by atorvastatin treatment, but was not changed significantly by simvastatin. Hemorrhage intensity per brain was commensurately decreased by Rock inhibition. Conclusions - These results support the exploration of proof of concept effect of high-dose atorvastatin on human CCM disease for potential therapeutic testing.
• Tung, E. L., Chua, R. F., Besser, S. A., Lindau, S. T., Kolak, M., Anyanwu, E. C., Liao, J. K., & Tabit, C. E. (2019). Association of Rising Violent Crime with Blood Pressure and Cardiovascular Risk: Longitudinal Evidence from Chicago, 2014-2016. American Journal of Hypertension, 32(Issue 12). doi:10.1093/ajh/hpz134
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  BACKGROUND: The purpose of this study was to examine the longitudinal association between rising violent crime and elevated blood pressure (BP). METHODS: We analyzed 217,816 BP measurements from 17,783 adults during a temporal surge in violent crime in Chicago (2014-2016). Serial observations were abstracted from the electronic health record at an academic medical center and paired to the City of Chicago Police Data Portal. The violent crime rate (VCR) was calculated as the number of violent crimes per 1,000 population per year for each census tract. Longitudinal multilevel regression models were implemented to assess elevated BP (systolic BP ≥ 140 mm Hg or diastolic BP ≥ 90 mm Hg) as a function of the VCR, adjusting for patient characteristics, neighborhood characteristics, and time effects. Secondary dependent measures included elevated heart rate, obesity, missed outpatient appointments, all-cause hospital admissions, and cardiovascular hospital admissions. RESULTS: At baseline, the median VCR was 41.3 (interquartile range: 15.2-66.8), with a maximum rise in VCR of 59.1 over the 3-year surge period. A 20-unit rise in the VCR was associated with 3% higher adjusted odds of having elevated BP (95% confidence interval [CI]: 1.01-1.06), 8% higher adjusted odds of missing an outpatient appointment (95% CI: 1.03-1.13), and 6% higher adjusted odds of having a cardiovascular-related hospital admission (95% CI: 1.01-1.12); associations were not significant for elevated heart rate and obesity. CONCLUSION: Rising violent crime was associated with increased BP during a temporal crime surge.
• Hiroi, Y., Noma, K., Kim, H. H., Sladojevic, N., Tabit, C. E., Li, Y., Soydan, G., Salomone, S., Moskowitz, M. A., & Liao, J. K. (2018). Neuroprotection mediated by upregulation of endothelial nitric oxide synthase in rho-associated, coiled-coil-containing kinase 2 deficient mice. Circulation Journal, 82(Issue 4). doi:10.1253/circj.cj-17-0732
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  Background: Rho-associated kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton and endothelial nitric oxide synthase (eNOS). Because the phosphorylation of eukaryotic elongation factor-1A1 (eEF1A1) by ROCK2 is critical for eNOS expression, we hypothesized that this molecular pathway may play a critical role in neuroprotection following focal cerebral ischemia. Methods and Results: Adult male wild-type (WT) and mutant ROCK2 and eNOS−/− mice were subjected to middle cerebral artery occlusion (MCAO), and cerebral infarct size, neurological deficit and absolute cerebral blood flow were measured. In addition, aortic endothelium-dependent response to acetylcholine, N G-nitro-L-arginine methyl ester (L-NAME) and sodium nitroprusside were assessed ex vivo. Endothelial cells from mouse brain or heart were used to measure eNOS and eEF1A activity, as well as NO production and eNOS mRNA half-life. In global hemizygous ROCK2+/− and endothelial-specific EC-ROCK2−/− mice, eNOS mRNA stability and eNOS expression were increased, which correlated with enhanced endothelium-dependent relaxation and neuroprotection following focal cerebral ischemia. Indeed, when ROCK2+/− mice were place on an eNOS−/− background, the neuroprotective effects observed in ROCK2+/− mice were abolished. Conclusions: These findings indicate that the phosphorylation of eEF1A1 by ROCK2 is physiologically important for eNOS expression and NO-mediated neuroprotection, and suggest that targeting endothelial ROCK2 and eEF1A may have therapeutic benefits in ischemic stroke and cardiovascular disease.
• Knipe, R. S., Probst, C. K., Lagares, D., Franklin, A., Spinney, J. J., Brazee, P. L., Grasberger, P., Zhang, L., Black, K. E., Sakai, N., Shea, B. S., Liao, J. K., Medoff, B. D., & Tager, A. M. (2018). The rho kinase isoforms ROCK1 and ROCK2 each contribute to the development of experimental pulmonary fibrosis. American Journal of Respiratory Cell and Molecular Biology, 58(Issue 4). doi:10.1165/rcmb.2017-0075oc
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  Pulmonary fibrosis is thought to result from dysregulated wound repair after repetitive lung injury. Many cellular responses to injury involve rearrangements of the actin cytoskeleton mediated by the two isoforms of the Rho-associated coiled-coil-forming protein kinase (ROCK), ROCK1 and ROCK2. In addition, profibrotic mediators such as transforming growth factor-b, thrombin, and lysophosphatidic acid act through receptors that activate ROCK. Inhibition of ROCK activation may be a potent therapeutic strategy for human pulmonary fibrosis. Pharmacological inhibition of ROCK using nonselective ROCK inhibitors has been shown to prevent fibrosis in animal models; however, the specific roles of each ROCK isoform are poorly understood. Furthermore, the pleiotropic effects of this kinase have raised concerns about on-target adverse effects of ROCK inhibition such as hypotension. Selective inhibition of one isoform might be a better-tolerated strategy. In the present study, we used a genetic approach to determine the roles of ROCK1 and ROCK2 in a mouse model of bleomycin-induced pulmonary fibrosis. Using ROCK1- or ROCK2-haploinsufficient mice, we found that reduced expression of either ROCK1 or ROCK2 was sufficient to protect them from bleomycin-induced pulmonary fibrosis. In addition, we found that both isoforms contribute to the profibrotic responses of epithelial cells, endothelial cells, and fibroblasts. Interestingly, ROCK1- and ROCK2-haploinsufficient mice exhibited similar protection from bleomycin-induced vascular leak, myofibroblast differentiation, and fibrosis; however, ROCK1-haploinsufficient mice demonstrated greater attenuation of epithelial cell apoptosis. These findings suggest that selective inhibition of either ROCK isoform has the potential to be an effective therapeutic strategy for pulmonary fibrosis.
• Li, S., Wang, Y., Jiang, Z., Huai, Y., Liao, J. K., Lynch, K. A., Zafonte, R., Wood, L. J., & Wang, Q. M. (2018). Impaired cognitive performance in endothelial nitric oxide synthase knockout mice after ischemic stroke. American Journal of Physical Medicine and Rehabilitation, 97(Issue 7). doi:10.1097/phm.0000000000000904
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  Objectives Cognitive dysfunction and dementia are common following ischemic stroke. Endothelial nitric oxide synthase (eNOS) has been found to play an important role in neurologic function and cognition. The purpose of the present study was to assess the specific role of eNOS in cognitive performance after stroke. Design Male wild-type and mice lacking eNOS (eNOS -/-) underwent middle cerebral artery occlusion or sham-surgery. Primary outcomes were repeated measures of neurologic score, limb asymmetry, sensory/motor function, and spatial memory/learning assessed at intervals up to 28 days postsurgery. Group differences in brain microglia activation and infiltration and levels of interferon-gamma were examined. Results There was no genotype × surgery interaction effect on the pattern of change in neurologic score, limb asymmetry, or sensory motor function across the 28 days postsurgery. In the Morris water maze, eNOS -/- middle cerebral artery occlusion mice displayed learning and memory deficits not evident in wild-type middle cerebral artery occlusion mice. Poorer spatial memory and learning in eNOS -/- middle cerebral artery occlusion mice was associated with a reduction in the number of activated microglia in the striatum on the lesion side and decreased brain tissue levels of interferon-gamma. Conclusions This study's data support a role for eNOS in cognitive performance after stroke. This finding may lead to the development of novel interventions to treat poststroke cognitive deficits.
• Tabit, C. E., Coplan, M. J., Chen, P., Jeevanandam, V., Uriel, N., & Liao, J. K. (2018). Tumor necrosis factor-α levels and non-surgical bleeding in continuous-flow left ventricular assist devices. Journal of Heart and Lung Transplantation, 37(Issue 1). doi:10.1016/j.healun.2017.06.001
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  Background Non-surgical bleeding (NSB) due to angiodysplasia is common in left ventricular assist device (LVAD) patients. Thrombin-induced angiopoietin-2 (Ang-2) expression in LVAD patients leads to altered angiogenesis and is associated with lower angiopoietin-1 (Ang-1) and increased NSB. However, the mechanism for decreased Ang-1, made by pericytes, is unknown and the origin of thrombin in LVAD patients is unclear. We hypothesized that high tumor necrosis factor-α (TNF-α) levels in LVAD patients induce pericyte apoptosis, tissue factor (TF) expression and vascular instability. Methods We incubated cultured pericytes with serum from patients with heart failure (HF), LVAD or orthotopic heart transplantation (OHT), with or without TNF-α blockade. We performed several measurements: Ang-1 expression was assessed by reverse transcript-polymerase chain reaction (RT-PCR) and pericyte death fluorescently; TF expression was assessed by RT-PCR in cultured endothelial cells incubated with patient plasma with or without TNF-α blockade; and TF expression was assessed in endothelial biopsy samples from these patients by immunofluorescence. We incubated cultured endothelial cells on Matrigel with patient serum with or without TNF-α blockade and determined tube formation by microscopy. Results Serum from LVAD patients had higher levels of TNF-α suppressed Ang-1 expression in pericytes, and induced pericyte death, and there was accelerated endothelial tube formation compared with serum from patients without LVADs. TF was higher in both plasma and endothelial cells from LVAD patients, and plasma from LVAD patients induced more endothelial TF expression. All of these effects were reversed or reduced with TNF-α blockade. High levels of TNF-α were associated with increased risk of NSB. Conclusions Elevated TNF-α in LVAD patients is a central regulator of altered angiogenesis, pericyte apoptosis and expression of TF and Ang-1.
• Kasahara, D. I., Mathews, J. A., Ninin, F. M., Wurmbrand, A. P., Liao, J. K., & Shore, S. A. (2017). Role of ROCK2 in CD4+ cells in allergic airways responses in mice. Clinical and Experimental Allergy, 47(Issue 2). doi:10.1111/cea.12866
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  Background: Rho kinases (ROCKs) contribute to allergic airways disease. ROCKs also play a role in lymphocyte proliferation and migration. Objective: To determine the role of ROCK2 acting within CD4+ cells in allergic airways responses. Methods: ROCK2-haploinsufficient (ROCK2+/−) and wild-type mice were sensitized with ovalbumin (OVA). ROCK2+/− mice then received either CD4+ cells from ROCK2-sufficient OVA TCR transgenic (OT-II) mice or saline i.v. 48 h before challenge with aerosolized OVA. Wild-type mice received saline before challenge. Allergic airways responses were measured 48 h after the last challenge. Allergic airways responses were also assessed in mice lacking ROCK2 only in CD4+ cells (ROCK2CD 4Cre mice) vs. control (CD4-Cre and ROCK2flox/flox) mice. Results: OVA-induced increases in bronchoalveolar lavage lymphocytes, eosinophils, IL-13, IL-5, and eotaxin were reduced in ROCK2+/− vs. wild-type mice, as were airway hyperresponsiveness and mucous hypersecretion. In ROCK2+/− mice, adoptive transfer with CD4+ cells from OT-II mice restored effects of OVA on lymphocytes, eosinophils, IL-13, IL-5, and mucous hypersecretion to wild-type levels, whereas eotaxin and airway hyperresponsiveness were not affected. ROCK2 inhibitors reduced IL-13-induced release of eotaxin from airway smooth muscle (ASM), similar to effects of these inhibitors on ASM contractility. Despite the ability of adoptive transfer to restore allergic airways inflammation in ROCK2-insufficient mice, allergic inflammation was not different in ROCK2CD 4Cre vs. control mice. Conclusion: ROCK2 contributes to allergic airways responses likely via effects within ASM cells and within non-lymphocyte cells involved in lymphocyte activation and migration into the airways.
• Shenkar, R., Shi, C., Austin, C., Moore, T., Lightle, R., Cao, Y., Zhang, L., Wu, M., Zeineddine, H. A., Girard, R., Mcdonald, D. A., Rorrer, A., Gallione, C., Pytel, P., Liao, J. K., Marchuk, D. A., & Awad, I. A. (2017). RhoA kinase inhibition with fasudil versus simvastatin in murine models of cerebral cavernous malformations. Stroke, 48(Issue 1). doi:10.1161/strokeaha.116.015013
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  Background and Purpose - We sought to compare the effect of chronic treatment with commonly tolerated doses of Fasudil, a specific RhoA kinase (ROCK) inhibitor, and simvastatin (with pleiotropic effects including ROCK inhibition) on cerebral cavernous malformation (CCM) genesis and maturation in 2 models that recapitulate the human disease. Methods - Two heterozygous murine models, Ccm1+/- Msh2 - /- and Ccm2+/- Trp53-/-, were treated from weaning to 4 to 5 months of age with Fasudil (100 mg/kg per day), simvastatin (40 mg/kg per day) or with placebo. Mouse brains were blindly assessed for CCM lesion burden, nonheme iron deposition (as a quantitative measure of chronic lesional hemorrhage), and ROCK activity. Results - Fasudil, but not simvastatin, significantly decreased mature CCM lesion burden in Ccm1+/- Msh2-/- mice, and in meta-analysis of both models combined, when compared with mice receiving placebo. Fasudil and simvastatin both significantly decreased the integrated iron density per mature lesion area in Ccm1+/- Msh2-/- mice, and in both models combined, compared with mice given placebo. ROCK activity in mature lesions of Ccm1+/- Msh2-/- mice was similar with both treatments. Fasudil, but not simvastatin, improved survival in Ccm1+/- Msh2-/- mice. Fasudil and simvastatin treatment did not affect survival or lesion development significantly in Ccm2+/- Trp53-/- mice alone, and Fasudil benefit seemed limited to males. Conclusions - ROCK inhibitor Fasudil was more efficacious than simvastatin in improving survival and blunting the development of mature CCM lesions. Both drugs significantly decreased chronic hemorrhage in CCM lesions. These findings justify the development of ROCK inhibitors and the clinical testing of commonly used statin agents in CCM.
• Shimizu, T., Narang, N., Chen, P., Yu, B., Knapp, M., Janardanan, J., Blair, J., & Liao, J. K. (2017). Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction. JCI Insight, 2(Issue 13). doi:10.1172/jci.insight.93187
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  Although left ventricular (LV) diastolic dysfunction is often associated with hypertension, little is known regarding its underlying pathophysiological mechanism. Here, we show that the actin cytoskeletal regulator, Rho-associated coiled-coil containing kinase-2 (ROCK2), is a critical mediator of LV diastolic dysfunction. In response to angiotensin II (Ang II), mutant mice with fibroblast-specific deletion of ROCK2 (ROCK2Postn–/–) developed less LV wall thickness and fibrosis, along with improved isovolumetric relaxation. This corresponded with decreased connective tissue growth factor (CTGF) and fibroblast growth factor–2 (FGF2) expression in the hearts of ROCK2Postn–/– mice. Indeed, knockdown of ROCK2 in cardiac fibroblasts leads to decreased expression of CTGF and secretion of FGF2, and cardiomyocytes incubated with conditioned media from ROCK2-knockdown cardiac fibroblasts exhibited less hypertrophic response. In contrast, mutant mice with elevated fibroblast ROCK activity exhibited enhanced Ang II–stimulated cardiac hypertrophy and fibrosis. Clinically, higher leukocyte ROCK2 activity was observed in patients with diastolic dysfunction compared with age- and sex-matched controls, and correlated with higher grades of diastolic dysfunction by echocardiography. These findings indicate that fibroblast ROCK2 is necessary to cause cardiac hypertrophy and fibrosis through the induction CTGF and FGF2, and they suggest that targeting ROCK2 may have therapeutic benefits in patients with LV diastolic dysfunction.
• Sladojevic, N., Taeg Oh, G., Kim, H. H., Beaulieu, L. M., Falet, H., Kamiński, K., Freedman, J. E., & Liao, J. K. (2017). Decreased thromboembolic stroke but not atherosclerosis or vascular remodelling in mice with ROCK2-deficient platelets. Cardiovascular Research, 113(Issue 11). doi:10.1093/cvr/cvx071
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  Aims Rho-associated coiled-coil containing kinase (ROCK)-2 is an important mediator of the actin cytoskeleton. Because changes in the actin cytoskeleton are critical for platelet function, we hypothesized that ROCK2 in platelets will play important role in thrombosis and can be potentially a target for therapeutic intervention in thromboembolic stroke. Methods and results We generated platelet-specific ROCK2-deficient mice (ROCK2Plt-/-) from conditional ROCK2fl_x/fl_x and platelet factor (PF)-4-Cre transgenic mice. Platelets from ROCK2Plt-/- mice were less responsive to thrombin stimulation in terms of pseudopodia formation, collagen adhesion, and in the formation of homotypic and heterotypic aggregates. This corresponded to prolonged bleeding time and delayed vascular occlusion following vessel injury. To determine whether these changes in platelet function could affect thrombotic disease, we utilized a clot-embolic model of ischaemic stroke. When pre-formed clots from ROCK2Plt-/- mice were injected into the middle cerebral artery of control mice, cerebral blood flow recovery occurred more rapidly, leading to decreased cerebral injury and neurological deficits, compared to pre-formed clots from control mice. Interestingly, pre-formed clots from control mice produced similar degree of cerebral injury when injected into control or ROCK2Plt-/- mice, suggesting that platelet ROCK2 deficiency affects clot formation but not propagation. Indeed, in a non-thrombotic intra-filament MCA occlusion model of stroke, platelet ROCK2 deletion was not protective. Furthermore, ROCK2Plt-/- mice exhibit similar atherosclerosis severity and vascular remodeling as control mice. Conclusion These findings indicate that platelet ROCK2 plays important role in platelet function and thrombosis, but does not contribute to the pathogenesis of atherosclerosis and vascular remodeling.
• Tabit, C. E., Coplan, M. J., Spencer, K. T., Alcain, C. F., Spiegel, T., Vohra, A. S., Adelman, D., Liao, J. K., & Sanghani, R. M. (2017). Cardiology Consultation in the Emergency Department Reduces Re-hospitalizations for Low-Socioeconomic Patients with Acute Decompensated Heart Failure. American Journal of Medicine, 130(Issue 9). doi:10.1016/j.amjmed.2017.03.044
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  Background Re-hospitalization after discharge for acute decompensated heart failure is a common problem. Low-socioeconomic urban patients suffer high rates of re-hospitalization and often over-utilize the emergency department (ED) for their care. We hypothesized that early consultation with a cardiologist in the ED can reduce re-hospitalization and health care costs for low-socioeconomic urban patients with acute decompensated heart failure. Methods There were 392 patients treated at our center for acute decompensated heart failure who received standardized education and follow-up. Patients who returned to the ED received early consultation with a cardiologist; 392 patients who received usual care served as controls. Thirty- and 90-day re-hospitalization, ED re-visits, heart failure symptoms, mortality, and health care costs were recorded. Results Despite guideline-based education and follow-up, the rate of ED re-visits was not different between the groups. However, the rate of re-hospitalization was significantly lower in patients receiving the intervention compared with controls (odds ratio 0.592), driven by a reduction in the risk of readmission from the ED (0.56 vs 0.79, respectively). Patients receiving the intervention accumulated 14% fewer re-hospitalized days than controls and 57% lower 30-day total health care cost. Despite the reduction in health care resource consumption, mortality was unchanged. After accounting for the total cost of intervention delivery, the health care cost savings was substantially greater than the cost of intervention delivery. Conclusion Early consultation with a cardiologist in the ED as an adjunct to guideline-based follow-up is associated with reduced re-hospitalization and health care cost for low-socioeconomic urban patients with acute decompensated heart failure.
• Girard, R., Khanna, O., Shenkar, R., Zhang, L., Wu, M., Jesselson, M., Zeineddine, H. A., Gangal, A., Fam, M. D., Gibson, C. C., Whitehead, K. J., Li, D. Y., Liao, J. K., Shi, C., & Awad, I. A. (2016). Peripheral plasma Vitamin D and non-HDL cholesterol reflect the severity of cerebral cavernous malformation disease. Biomarkers in Medicine, 10(Issue 3). doi:10.2217/bmm.15.118
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  Aim: To correlate cerebral cavernous malformations (CCMs) disease aggressiveness with peripheral blood biomarkers hypothesized mechanistically. Patients & methods: A prospective case-control study enrolled 43 CCM patients, where 25-(OH) vitamin D, HDL and non-HDL cholesterol, CRP plasma levels and leukocyte ROCK activity were correlated with parameters of disease aggressiveness reflecting chronic and acute domains. Results: Patients with one or more features of chronically aggressive disease (early age at symptom onset, two or more symptomatic bleeds, high lesion burden) had significantly lower 25-(OH) vitamin D and non-HDL cholesterol levels in comparison to patients without these features. Conclusion: Validation of these biomarkers and their potential treatment modulation may influence the clinical care of patients with CCM disease.
• Hofmann Bowman, M. A., & Liao, J. K. (2016). Relative lack of culprit and obstructive coronary lesions in patients with acute ischemic stroke and elevated cardiac troponin. Circulation, 133(Issue 13). doi:10.1161/circulationaha.116.021795
• Lennon, F. E., Cianci, G. C., Kanteti, R., Riehm, J. J., Arif, Q., Poroyko, V. A., Lupovitch, E., Vigneswaran, W., Husain, A., Chen, P., Liao, J. K., Sattler, M., Kindler, H. L., & Salgia, R. (2016). Unique fractal evaluation and therapeutic implications of mitochondrial morphology in malignant mesothelioma. Scientific Reports, 6(Issue). doi:10.1038/srep24578
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  Malignant mesothelioma (MM), is an intractable disease with limited therapeutic options and grim survival rates. Altered metabolic and mitochondrial functions are hallmarks of MM and most other cancers. Mitochondria exist as a dynamic network, playing a central role in cellular metabolism. MM cell lines display a spectrum of altered mitochondrial morphologies and function compared to control mesothelial cells. Fractal dimension and lacunarity measurements are a sensitive and objective method to quantify mitochondrial morphology and most importantly are a promising predictor of response to mitochondrial inhibition. Control cells have high fractal dimension and low lacunarity and are relatively insensitive to mitochondrial inhibition. MM cells exhibit a spectrum of sensitivities to mitochondrial inhibitors. Low mitochondrial fractal dimension and high lacunarity correlates with increased sensitivity to the mitochondrial inhibitor metformin. Lacunarity also correlates with sensitivity to Mdivi-1, a mitochondrial fission inhibitor. MM and control cells have similar sensitivities to cisplatin, a chemotherapeutic agent used in the treatment of MM. Neither oxidative phosphorylation nor glycolytic activity, correlated with sensitivity to either metformin or mdivi-1. Our results suggest that mitochondrial inhibition may be an effective and selective therapeutic strategy in mesothelioma, and identifies mitochondrial morphology as a possible predictor of response to targeted mitochondrial inhibition.
• Tabit, C. E., Chen, P., Kim, G. H., Fedson, S. E., Sayer, G., Coplan, M. J., Jeevanandam, V., Uriel, N., & Liao, J. K. (2016). Elevated Angiopoietin-2 Level in Patients with Continuous-Flow Left Ventricular Assist Devices Leads to Altered Angiogenesis and Is Associated with Higher Nonsurgical Bleeding. Circulation, 134(Issue 2). doi:10.1161/circulationaha.115.019692
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  Background: Nonsurgical bleeding is the most common adverse event in patients with continuous-flow left ventricular assist devices (LVADs) and is caused by arteriovenous malformations. We hypothesized that deregulation of an angiogenic factor, angiopoietin-2 (Ang-2), in patients with LVADs leads to increased angiogenesis and higher nonsurgical bleeding. Methods: Ang-2 and thrombin levels were measured by ELISA and Western blotting, respectively, in blood samples from 101 patients with heart failure, LVAD, or orthotopic heart transplantation. Ang-2 expression in endothelial biopsy was quantified by immunofluorescence. Angiogenesis was determined by in vitro tube formation from serum from each patient with or without Ang-2-blocking antibody. Ang-2 gene expression was measured by reverse transcription-polymerase chain reaction in endothelial cells incubated with plasma from each patient with or without the thrombin receptor blocker vorapaxar. Results: Compared with patients with heart failure or those with orthotopic heart transplantation, serum levels and endothelial expression of Ang-2 were higher in LVAD patients (P=0.001 and P
• Zhou, Q., Einert, M., Schmitt, H., Wang, Z., Pankratz, F., Olivier, C. B., Bode, C., Liao, J. K., & Moser, M. (2016). MnTBAP increases BMPR-II expression in endothelial cells and attenuates vascular inflammation. Vascular Pharmacology, 84(Issue). doi:10.1016/j.vph.2016.07.001
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  Aims The endothelium plays an important role during vascular inflammation. Previous data have demonstrated a high expression level of manganese-superoxide dismutase (MnSOD) in endothelial cells and suggested an important role of MnSOD in several cardiovascular diseases. Manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) has been shown to mimic some of the effects of MnSOD and prevented the development of diabetes and obesity. However, its effect on vascular inflammation and the underlying mechanism is still unknown. Methods and results Leukocyte adhesion was evaluated in-vivo and in-vitro using dynamic flow chamber and intravital microscopy in mice. Expression of adhesion molecules induced by TNFα and adhesion of leukocytes to the vessel wall were inhibited by MnTBAP. The anti-inflammatory effect of MnTBAP was partly mediated by up-regulation of the BMPR-II and Smad dependent pathway. Additionally, MnTBAP decelerated the turn-over of endogenous BMPR-II. Conclusion Our data demonstrate that MnTBAP activates Smad signaling, preserves the turn-over of BMPR-II and elicits anti-inflammatory effects in endothelial cells, partly mediated by BMPR-II. This finding suggests a potential therapeutic impact of MnTBAP in the treatment of vascular inflammation.
• Hsieh, M. L., Liu, P. Y., Wu, J. M., Liao, J. K., & Wang, J. N. (2015). Interventional transcatheter closure ameliorates the leukocyte rho kinase activities among patients with patent ductus arteriosus. Acta Cardiologica Sinica, 31(Issue 6). doi:10.6515/acs20150424d
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  Background: Patent ductus arteriosus (PDA) causes increased pulmonary blood flow, which can lead to pulmonary arterial hypertension (PAH). Rho-associated coiled-coil containing protein kinase (ROCK) may play an important pathophysiological role in PAH. We hypothesized that the increased pulmonary artery (PA) flow from PDA could activate ROCK. Methods: Patients who received a PDA transcatheter closure in our hospital were consecutively enrolled in this study. Basic demographics and clinical hemodynamic data of the study participants were recorded. Then, ROCK activity was measured before and after the PDA occlusion procedure. ROCK activity was defined as the phosphorylation ratio ofmyosin-binding subunit byWestern blot measurement.We also sub-divided patients into the coil group and occluder group based on the occlusion device used in each patient's procedure. Results: From January 2009 to December 2011, 25 patients with a median age of 2.3 years, ranging from 10 months to 72 years were enrolled. The mean PDA size was 0.31 ± 0.14 cm, the mean Qp/Qs shunt was 1.54 ± 0.41, and the mean systolic pulmonary artery pressure was 26.9 ± 10.3 mmHg. There were 10 patients (one boy and nine girls) in the coil group and 15 patients (four boys and eleven girls) in the occluder group. Following the closure of the PDA, ROCK activity significantly decreased (1.78 ± 2.25 vs. 0.77 ± 0.69, p > 0.01). There was a strong correlation between the leukocyte ROCK activity with the systolic PA pressure (y = 5.4608x + 22.54, R2 = 0.5539, p > 0.05), but not the Qp/Qs value. Both subgroups showed significant changes of ROCK activity after the procedure. Interestingly, when comparing the coil group with the occluder group, the decrease in ROCK activity was more apparent in the occluder group. Conclusions: The findings of this study indicated that ROCK activity is higher in patients with PDA and correlates with PA pressure. The decrease in ROCK activity following the device closure suggests that ROCK may be an important biomarker for PDA patency.
• Kajikawa, M., Noma, K., Nakashima, A., Maruhashi, T., Iwamoto, Y., Matsumoto, T., Iwamoto, A., Oda, N., Hidaka, T., Kihara, Y., Aibara, Y., Chayama, K., Sasaki, S., Kato, M., Dote, K., Goto, C., Liao, J. K., & Higashi, Y. (2015). Rho-associated kinase activity is an independent predictor of cardiovascular events in acute coronary syndrome. Hypertension, 66(Issue 4). doi:10.1161/hypertensionaha.115.05587
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  Rho-associated kinases play an important role in a variety of cellular functions. Although Rho-associated kinase activity has been shown to be an independent predictor for future cardiovascular events in a general population, there is no information on Rho-associated kinase activity in patients with acute coronary syndrome. We evaluated leukocyte Rho-associated kinase activity by Western blot analysis in 73 patients with acute coronary syndrome and 73 age- and gender-matched control subjects. Rho-associated kinase activity within 2 hours of acute coronary syndrome onset was higher in patients with acute coronary syndrome than in the control subjects (0.95±0.55 versus 0.69±0.31; P
• Kasahara, D. I., Mathews, J. A., Park, C. Y., Cho, Y., Hunt, G., Wurmbrand, A. P., Liao, J. K., & Shore, S. A. (2015). ROCK insufficiency attenuates ozone-induced airway hyperresponsiveness in mice. American Journal of Physiology - Lung Cellular and Molecular Physiology, 309(Issue 7). doi:10.1152/ajplung.00372.2014
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  Ozone causes airway hyperresponsiveness (AHR) and pulmonary inflammation. Rho kinase (ROCK) is a key regulator of smooth muscle cell contraction and inflammatory cell migration. To determine the contribution of the two ROCK isoforms ROCK1 and ROCK2 to ozone-induced AHR, we exposed wild-type, ROCK1+/-, and ROCK2+/- mice to air or ozone (2 ppm for 3 h) and evaluated mice 24 h later. ROCK1 or ROCK2 haploinsufficiency did not affect airway responsiveness in air-exposed mice but significantly reduced ozone-induced AHR, with a greater reduction in ROCK2+/- mice despite increased bronchoalveolar lavage (BAL) inflammatory cells in ROCK2+/- mice. Compared with wild-type mice, ozone-induced increases in BAL hyaluronan, a matrix protein implicated in ozone-induced AHR, were lower in ROCK1+/- but not ROCK2+/- mice. Ozone-induced increases in other inflammatory moieties reported to contribute to ozone-induced AHR (IL-17A, osteopontin, TNF_) were not different in wild-type vs. ROCK1+/- or ROCK2+/- mice. We also observed a dose-dependent reduction in ozone-induced AHR after treatment with the ROCK1/ ROCK2 inhibitor fasudil, even though fasudil was administered after induction of inflammation. Ozone increased pulmonary expression of ROCK2 but not ROCK1 or RhoA. A ROCK2 inhibitor, SR3677, reduced contractile forces in primary human airway smooth muscle cells, confirming a role for ROCK2 in airway smooth muscle contraction. Our results demonstrate that ozone-induced AHR requires ROCK. Whereas ROCK1-dependent changes in hyaluronan may contribute to ROCK1’s role in O3-induced AHR, the role of ROCK2 is downstream of inflammation, likely at the level of airway smooth muscle contraction.
• Kasahara, D. I., Ninin, F. M., Wurmbrand, A. P., Liao, J. K., & Shore, S. A. (2015). Abrogation of airway hyperresponsiveness but not inflammation by rho kinase insufficiency. Clinical and Experimental Allergy, 45(Issue 2). doi:10.1111/cea.12438
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  Summary: Background: Major features of allergic asthma include airway hyperresponsiveness (AHR), eosinophilic inflammation, and goblet cell metaplasia. Rho kinase (ROCK) is a serine/threonine protein kinase that regulates the actin cytoskeleton. By doing so, it can modulate airway smooth muscle cell contraction and leucocyte migration and proliferation. This study was designed to determine the contributions of the two ROCK isoforms, ROCK1 and ROCK2, to AHR, inflammation and goblet cell metaplasia in a mast cell-dependent model of allergic airways disease. Methods and Results: Repeated intranasal challenges with OVA caused AHR, eosinophilic inflammation, and goblet cell hyperplasia in wild-type (WT) mice. OVA-induced AHR was partially or completely abrogated in mice haploinsufficient for ROCK2 (ROCK2+/-) or ROCK1 (ROCK1+/-), respectively. In contrast, there was no effect of ROCK insufficiency on allergic airways inflammation, although both ROCK1 and ROCK2 insufficiency attenuated mast cell degranulation. Goblet cell hyperplasia, as indicated by PAS staining, was not different in ROCK1+/- vs. WT mice. However, in ROCK2+/- mice, goblet cell hyperplasia was reduced in medium but not large airways. Maximal acetylcholine-induced force generation was reduced in tracheal rings from ROCK1+/- and ROCK2+/- vs. WT mice. The ROCK inhibitor, fasudil, also reduced airway responsiveness in OVA-challenged mice, without affecting inflammatory responses. Conclusion: In a mast cell model of allergic airways disease, ROCK1 and ROCK2 both contribute to AHR, likely through direct effects on smooth muscle cell and effects on mast cell degranulation. In addition, ROCK2 but not ROCK1 plays a role in allergen-induced goblet cell hyperplasia.
• Knipe, R. S., Tager, A. M., & Liao, J. K. (2015). The rho kinases: Critical mediators of multiple profibrotic processes and rational targets for new therapies for pulmonary fibrosis. Pharmacological Reviews, 67(Issue 1). doi:10.1124/pr.114.009381
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  Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung scarring, short median survival, and limited therapeutic options, creating great need for new pharmacologic therapies. IPF is thought to result from repetitive environmental injury to the lung epithelium, in the context of aberrant host wound healing responses. Tissue responses to injury fundamentally involve reorganization of the actin cytoskeleton of participating cells, including epithelial cells, fibroblasts, endothelial cells, and macrophages. Actin filament assembly and actomyosin contraction are directed by the Rho-associated coiledcoil forming protein kinase (ROCK) family of serine/ threonine kinases (ROCK1 and ROCK2). As would therefore be expected, lung ROCK activation has been demonstrated in humans with IPF and in animal models of this disease. ROCK inhibitors can prevent fibrosis in these models, and more importantly, induce the regression of already established fibrosis. Here we review ROCK structure and function, upstream activators and downstream targets of ROCKs in pulmonary fibrosis, contributions of ROCKs to profibrotic cellular responses to lung injury, ROCK inhibitors and their efficacy in animal models of pulmonary fibrosis, and potential toxicities of ROCK inhibitors in humans, as well as involvement of ROCKs in fibrosis in other organs. As we discuss, ROCK activation is required for multiple profibrotic responses, in the lung and multiple other organs, suggesting ROCK participation in fundamental pathways that contribute to the pathogenesis of a broad array of fibrotic diseases. Multiple lines of evidence therefore indicate that ROCK inhibition has great potential to be a powerful therapeutic tool in the treatment of fibrosis, both in the lung and beyond.
• Liao, Y. C., Liu, P. Y., Lin, H. F., Lin, W. Y., Liao, J. K., & Juo, S. H. (2015). Two functional polymorphisms of ROCK2 enhance arterial stiffening through inhibiting its activity and expression. Journal of Molecular and Cellular Cardiology, 79(Issue). doi:10.1016/j.yjmcc.2014.11.023
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  Derangement of Rho-associated kinases (ROCKs) has been related to coronary artery disease and stroke. ROCK2, rather than ROCK1, plays a predominant role in vascular contractility. The present study aims to test (1) the associations between ROCK2 single nucleotide polymorphisms (SNPs) and arterial stiffness, and (2) the molecular mechanism accounting for their effects. Stiffness parameters including beta (β), elasticity modulus (Ep) and pulse wave velocity (PWV) were obtained by carotid ultrasonography. Seven tagging SNPs of ROCK2 were initially genotyped in 856 subjects and significant SNPs were replicated in another group of 527 subjects. Two SNPs in complete linkage disequilibrium were found to be significantly associated with arterial stiffness. The major alleles of rs978906 (A allele) and rs9808232 (C allele) were associated with stiffer arteries. SNP rs978906 was predicted to influence microRNA(miR)-1183 binding to ROCK2, while rs9808232 causes amino acid substitution. To determine their functional impact, plasmid constructs carrying different alleles of the significant SNPs were created. Compared to rs978906G-allele constructs, cells transfected with rs978906A-allele constructs had higher baseline luciferase activities and were less responsive to miR-1183 changes. Oxidized-low density lipoprotein (Ox-LDL) suppressed miR-1183 levels and increased ROCK2 protein amounts. For rs9808232, cells transfected with C-allele constructs had significantly higher ROCK activities than those with A-allele constructs. Leukocyte ROCK activities were further measured in 52 healthy subjects. The average ROCK activity was highest in human subjects with CC genotype at rs9808232, followed by those with AC and lowest in AA. Taken together, the present study showed that two functional SNPs of ROCK2 increase susceptibility of arterial stiffness in the Chinese population. Non-synonymous SNP rs9808232 influences ROCK2 activity, while 3' UTR SNP rs978906 affects the ROCK2 protein synthesis by interfering miR-1183 binding.
• Maruhashi, T., Noma, K., Fujimura, N., Kajikawa, M., Matsumoto, T., Hidaka, T., Nakashima, A., Kihara, Y., Liao, J. K., & Higashi, Y. (2015). Exogenous nitric oxide inhibits Rho-associated kinase activity in patients with angina pectoris: A randomized controlled trial. Hypertension Research, 38(Issue 7). doi:10.1038/hr.2015.24
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  The RhoA/Rho-associated kinase (ROCK) pathway has a key physiological role in the pathogenesis of atherosclerosis. Increased ROCK activity is associated with cardiovascular diseases. Endogenous nitric oxide (NO) has an anti-atherosclerotic effect, whereas the exogenous NO-mediated cardiovascular effect still remains controversial. The purpose of this study was to evaluate the effect of exogenous NO on ROCK activity in patients with angina pectoris. This is a prospective, open-label, randomized, controlled study. A total of 30 patients with angina pectoris were randomly assigned to receive 40 mg day -1 of isosorbide mononitrate (n=15, 12 men and 3 women, mean age of 63±12 years, isosorbide mononitrate group) or conventional treatment (n=15, 13 men and 2 women, mean age of 64±13 years, control group) for 12 weeks. ROCK activity in peripheral leukocytes was measured by western blot analysis. ROCK activities at 4 and 12 weeks after treatment were decreased in the isosorbide mononitrate group (0.82±0.33 at 0 week, 0.62±0.20 at 4 weeks, 0.61±0.19 at 12 weeks, n=15 in each group, P
• Matsumoto, T., Oki, K., Kajikawa, M., Nakashima, A., Maruhashi, T., Iwamoto, Y., Iwamoto, A., Oda, N., Hidaka, T., Kihara, Y., Kohno, N., Chayama, K., Goto, C., Aibara, Y., Noma, K., Liao, J. K., & Higashi, Y. (2015). Effect of aldosterone-producing adenoma on endothelial function and rho-associated kinase activity in patients with primary aldosteronism. Hypertension, 65(Issue 4). doi:10.1161/hypertensionaha.114.05001
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  The purpose of this study was to evaluate vascular function and activity of Rho-associated kinases (ROCKs) in patients with primary aldosteronism. Vascular function, including flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation, and ROCK activity in peripheral leukocytes were evaluated in 21 patients with aldosterone-producing adenoma (APA), 23 patients with idiopathic hyperaldosteronism (IHA), and 40 age-, sex-, and blood pressure-matched patients with essential hypertension (EHT). FMD was significantly lower in the APA group than in the IHA and EHT groups (3.2±2.0% versus 4.6±2.3% and 4.4±2.2%; P
• Zhou, Q., Gensch, C., Keller, C., Schmitt, H., Esser, J., Moser, M., & Liao, J. K. (2015). MnTBAP stimulates angiogenic functions in endothelial cells through mitofusin-1. Vascular Pharmacology, 72(Issue). doi:10.1016/j.vph.2015.05.007
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  Aims: Angiogenesis is defined as the sprouting of capillaries from pre-existing vasculature. It is a complex process that includes endothelial proliferation, migration, and tube formation. Previous data have demonstrated a high expression level of manganese-superoxide dismutase (MnSOD) in endothelial cells and suggested an important role of MnSOD in several cardiovascular diseases. In addition, manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) has been shown to mimic some of the effects of MnSOD in various tissues. However, its effect on the vasculature remains unknown. Methods and results: HUVECs were treated with MnTBAP. Migration, tube formation, and capillary sprouting assays were performed to evaluate the pro-angiogenic effect in vitro. Matrigel plug assay was performed to assess capillary ingrowth in vivo. Compared to control, treatment with MnTBAP revealed increased cell migration, tube formation and capillary sprouting along with more capillary ingrowth in the Matrigel plug assay. This effect was mediated through a mitofusin (Mfn)-1-dependent pathway. Expression of Tie-2, Ang-2 and VEGF mRNA was increased in muscle tissues after ligation in MnTBAP treated mice. However, revascularization in the hindlimb ischemia model was not statistically significant at day 10 in MnTBAP treated mice. Conclusion: In summary, our data demonstrate a strong pro-angiogenic, but less pro-arteriogenic effect of MnTBAP in HUVECs mediated by Mfn-1.
• Kajikawa, M., Noma, K., Maruhashi, T., Mikami, S., Iwamoto, Y., Iwamoto, A., Matsumoto, T., Hidaka, T., Kihara, Y., Chayama, K., Nakashima, A., Goto, C., Liao, J. K., & Higashi, Y. (2014). Rho-associated kinase activity is a predictor of cardiovascular outcomes. Hypertension, 63(Issue 4). doi:10.1161/hypertensionaha.113.02296
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  Cardiovascular diseases are associated with chronic activation of Rho-associated kinase. Rho-associated kinase activity is significantly correlated with endothelial function and Framingham risk score. However, there is no information on the prognostic value of Rho-associated kinase activity. We evaluated Rho-associated kinase activity in peripheral leukocytes by Western blot analysis in 633 subjects who underwent health-screening examination at Hiroshima University Hospital. We assessed the associations between Rho-associated kinase activity and first major cardiovascular events (death from cardiovascular causes, myocardial infarction, and stroke), death from cardiovascular causes, acute myocardial infarction, stroke, revascularization (percutaneous coronary intervention, coronary artery bypass grafting), and hospitalization for heart failure. During a median period of 42.0 months (interquartile range, 24.4-56.6 months) of follow-up, 29 subjects died (10 from cardiovascular causes), 2 myocardial infarction, 20 revascularization, 15 stroke, and 17 hospitalization for heart failure. After adjustment for age, sex, cardiovascular risk factors, and other relevant variables, Rho-associated kinase activity remained a strong independent indicator of first major cardiovascular events (hazard ratio, 2.19; 95% confidence interval, 1.35-3.70; P=0.002), death from cardiovascular disease (hazard ratio, 2.57; 95% confidence interval, 1.18-6.60; P=0.002), stroke (hazard ratio, 2.14; 95% confidence interval, 1.24-3.86; P=0.006), and revascularization (hazard ratio, 2.68; 95% confidence interval, 1.60-4.66; P
• Liu, P. Y., Lee, P. T., Chang, W. T., Tai, Y. L., Chao, T. H., Lee, C. H., Li, Y. H., Chen, J. H., Tsai, L. M., & Liao, J. K. (2014). Evidence of pleiotropy by statins: Leukocyte Rho kinase (ROCK) activity and pretreated statin before percutaneous coronary interventions are clinical vascular outcome predictors. International Journal of Cardiology, 176(Issue 1). doi:10.1016/j.ijcard.2014.06.059
• Maruhashi, T., Noma, K., Iwamoto, Y., Iwamoto, A., Oda, N., Kajikawa, M., Matsumoto, T., Hidaka, T., Kihara, Y., Chayama, K., Nakashima, A., Goto, C., Liao, J. K., & Higashi, Y. (2014). Critical role of exogenous nitric oxide in ROCK activity in vascular smooth muscle cells. PLoS ONE, 9(Issue 10). doi:10.1371/journal.pone.0109017
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  Objective: Rho-associated kinase (ROCK) signaling pathway has been shown to mediate various cellular functions including cell proliferation, migration, adhesion, apoptosis, and contraction, all of which may be involved in pathogenesis of atherosclerosis. Endogenous nitric oxide (NO) is well known to have an anti-atherosclerotic effect, whereas the exogenous NO-mediated cardiovascular effect still remains controversial. The purpose of this study was to evaluate the effect of exogenous NO on ROCK activity in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Methods: VSMCs migration was evaluated using a modified Boyden chamber assay. ROCK activities were measured by Western blot analysis in murine and human VSMCs and aorta of mice treated with or without angiotensin II (Ang II) and/or sodium nitroprusside (SNP), an NO donor. Results: Co-treatment with SNP inhibited the Ang II-induced cell migration and increases in ROCK activity in murine and human VSMCs. Similarly, the increased ROCK activity 2 weeks after Ang II infusion in the mouse aorta was substantially inhibited by subcutaneous injection of SNP. Conclusions: These findings suggest that administration of exogenous NO can inhibit ROCK activity in VSMCs in vitro and in vivo.
• Zee, R. Y., Wang, Q. M., Chasman, D. I., Ridker, P. M., & Liao, J. K. (2014). Gene variations of ROCKs and risk of ischaemic stroke: The women's genome health study. Clinical Science, 126(Issue 12). doi:10.1042/cs20130652
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  Recent animal and human studies have demonstrated the importance of the ROCK (RhoA/Rho-associated kinase) pathway in IsST (ischaemic stroke). Whether the genetic variation within ROCK-associated genes modulates the risk of IsST remains elusive. The association between 66 tSNPs [tagging SNPs (single nucleotide polymorphisms)] of three ROCK-associated genes [ROCK1, ROCK2 and ARHGEF10 (Rho guanine-nucleotide-exchange factor 10)] and the incidence of IsST was investigated in 23294 Caucasian female participants of the prospective WGHS (Women's Genome Health Study). All were free of known cancer and cardiovascular disease at baseline. During a 15-year follow-up period, 323 participants developed their first ever IsST. Multivariable Cox regression analysis was performed to investigate the relationship between genotypes and risk of IsST assuming an additive genetic model. Haplotype-block analysis was also performed. A total of ten tSNPs were associated with the risk of IsST (three in ARHGEF10 and seven in ROCK1; P
• Dong, M., Jiang, X., Liao, J. K., & Yan, B. P. (2013). Elevated Rho-kinase activity as a marker indicating atherosclerosis and inflammation burden in polyvascular disease patients with concomitant coronary and peripheral arterial disease. Clinical Cardiology, 36(Issue 6). doi:10.1002/clc.22118
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  Background Recent evidence suggests that Rho-kinase (ROCK) plays an important role in the pathogenesis of atherosclerosis and a marker of atherosclerotic burden. Polyvascular disease with concomitant peripheral arterial disease (PAD) and coronary artery disease (CAD) is common and associated with a worse prognosis. The aim of this study was to evaluate ROCK activity as a marker of polyvascular disease. Hypothesis Methods We retrospectively analyzed patients undergoing coronary angiography at our institution between February 2009 and May 2009. Patients with only CAD (n = 40) defined by coronary artery stenosis of ≥50% by angiography, only PAD (n = 40) defined by an ankle brachial index (ABI)
• Dong, M., Liao, J. K., Yan, B., Li, R., Zhang, M., & Yu, C. M. (2013). A combination of increased Rho kinase activity and N-terminal pro-B-type natriuretic peptide predicts worse cardiovascular outcome in patients with acute coronary syndrome. International Journal of Cardiology, 167(Issue 6). doi:10.1016/j.ijcard.2012.07.007
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  Background: Recent experimental evidence suggests that the Rho/Rho-kinase (ROCK) system may play an important role in the pathogenesis of acute coronary syndrome (ACS) but there are little clinical data. This study examined if ROCK activity is increased in patients with acute coronary syndrome and if ROCK activity predicts long-term cardiovascular event. Method: Blood samples were collected from 188 patients within 12 h after admission for ACS (53% men; aged 70 ± 13) and from 61 control subject. The main outcome measures were all cause mortality, readmission with ACS or congestive heart failure (CHF) from presentation within around 2 years (mean:14.4 ± 7.2 months; range: 0.5 to 26 months). Results: ROCK activity increased in ST elevation myocardial infarction (STEMI, n = 90) (3.33 ± 0.93), non-STEMI (NSTEMI, n = 68) (3.37 ± 1.04) and unstable angina (UA, n = 30) (2.53 ± 0.59) groups when compared with disease controls (n = 31) (2.06 ± 0.38, all p < 0.001) and healthy controls (n = 30) (1.54 ± 0.43, all p < 0.001). There were 24 deaths, 34 readmissions with ACS and 15 admissions with CHF within 2 years. Patients with a high N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high ROCK activity on admission had a five-fold risk of a cardiovascular event (RR: 5.156; 95% CI: 2.180-12.191) when compared to those with low NT-proBNP and low ROCK activity. Conclusion: ROCK activity was increased in patients with ACS, particularly in those with myocardial infarction. The combined usage of both ROCK activity and NT-proBNP might identify a subset of ACS patients at particularly high risk. © 2012 Elsevier Ireland Ltd.
• Liao, J. K. (2013). Linking endothelial dysfunction with endothelial cell activation. Journal of Clinical Investigation, 123(Issue 2). doi:10.1172/jci66843
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  The thin layer of cells that lines the interior of blood vessels, known as the endothelium, plays a complex role in vascular biology. The endothelium mediates blood vessel tone, hemostasis, neutrophil recruitment, hormone trafficking, and fluid filtration. Endothelial dysfunction, as defined by a lack of NO, has been linked to a variety of disease states, including atherosclerosis, diabetes mellitus, coronary artery disease, hypertension, and hypercholesterolemia. Indeed, restoration of endothelial function is one of the earliest recognizable benefits of statin therapy. In 1995, James Liao and colleagues published a study in the JCI demonstrating that NO is a vascular protective factor that limits endothelial activation and prevents leukocyte adhesion to the vessel wall.
• Montalvo, J., Spencer, C., Hackathorn, A., Masterjohn, K., Perkins, A., Doty, C., Arumugam, A., Ongusaha, P. P., Lakshmanaswamy, R., Liao, J. K., Mitchell, D. C., & Bryan, B. A. (2013). Rock1 & 2 perform overlapping and unique roles in angiogenesis and angiosarcoma tumor progression. Current Molecular Medicine, 13(Issue 1). doi:10.2174/156652413804486296
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  The serine/threonine protein kinase paralogs ROCK1 & 2 have been implicated as essential modulators of angiogenesis; however their paralog-specific roles in endothelial function are unknown. shRNA knockdown of ROCK1 or 2 in endothelial cells resulted in a significant disruption of in vitro capillary network formation, cell polarization, and cell migration compared to cells harboring non-targeting control shRNA plasmids. Knockdowns led to alterations in cytoskeletal dynamics due to ROCK1 & 2-mediated reductions in actin isoform expression, and ROCK2-specific reduction in myosin phosphatase and cofilin phosphorylation. Knockdowns enhanced cell survival and led to ROCK1 & 2-mediated reduction in caspase 6 and 9 cleavage, and a ROCK2-specific reduction in caspase 3 cleavage. Microarray analysis of ROCK knockdown lines revealed overlapping and unique control of global transcription by the paralogs, and a reduction in the transcriptional regulation of just under 50% of VEGF responsive genes. Finally, paralog knockdown in xenograft angiosarcoma tumors resulted in a significant reduction in tumor formation. Our data reveals that ROCK1 & 2 exhibit overlapping and unique roles in normal and dysfunctional endothelial cells, that alterations in cytoskeletal dynamics are capable of overriding mitogen activated transcription, and that therapeutic targeting of ROCK signaling may have profound impacts for targeting angiogenesis.
• Okamoto, R., Li, Y., Noma, K., Hiroi, Y., Liu, P. Y., Taniguchi, M., Ito, M., & Liao, J. K. (2013). FHL2 prevents cardiac hypertrophy in mice with cardiac-specific deletion of ROCK2. FASEB Journal, 27(Issue 4). doi:10.1096/fj.12-217018
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  The Rho-associated coiled-coil containing kinases, ROCK1 and ROCK2, are important regulators of cell shape, migration, and proliferation through effects on the actin cytoskeleton. However, it is not known whether ROCK2 plays an important role in the development of cardiac hypertrophy. To determine whether the loss of ROCK2 could prevent cardiac hypertrophy, cardiomyocytespecific ROCK2-null (c-ROCK2-/-) were generated using conditional ROCK2 flox/flox mice and myosin heavy-chain promoter-driven Cre recombinase transgenic mice. Cardiac hypertrophy was induced by Ang II infusion (400 ng/kg/ min, 28 d) or transverse aortic constriction (TAC). Under basal conditions, hemodynamic parameters, cardiac anatomy, and function of c-ROCK2-/- mice were comparable to wild-type (WT) mice. However, following Ang II infusion or TAC, c-ROCK2-/- mice exhibited a substantially smaller increase in heart-to-body weight ratio, left ventricular mass, myocyte cross-sectional area, hypertrophy-related fetal gene expression, intraventricular fibrosis, cardiac apoptosis, and oxidative stress compared to control mice. Deletion of ROCK2 in cardiomyocytes leads to increased expression of four-and-a-half LIM-only protein-2 (FHL2) and FHL2- mediated inhibition of serum response factor (SRF) and extracellular signal-regulated mitogen-activated protein kinase (ERK). Knockdown of FHL2 expression in ROCK2-deficient cardiomyocytes or placing ROCK2-haploinsufficient (ROCK2+/-) mice on FHL2 +/-- haploinsufficient background restored the hypertrophic response to Ang II. These results indicate that cardiomyocyte ROCK2 is essential for the development of cardiac hypertrophy and that up-regulation of FHL2 may contribute to the antihypertrophic phenotype that is observed in cardiacspecific ROCK2-deficient mice.-Okamoto, R., Li, Y., Noma, K., Hiroi, Y., Liu, P.-Y., Taniguchi, M., Ito, M., Liao, J. K. FHL2 prevents cardiac hypertrophy in mice with cardiac-specific deletion of ROCK2. FASEB J. 27, 1439-1449 (2013). www.fasebj.org.
• Tanaka, S. I., Fukumoto, Y., Nochioka, K., Minami, T., Kudo, S., Shiba, N., Takai, Y., Williams, C. L., Liao, J. K., & Shimokawa, H. (2013). Statins exert the pleiotropic effects through small gtp-binding protein dissociation stimulator upregulation with a resultant rac1 degradation. Arteriosclerosis, Thrombosis, and Vascular Biology, 33(Issue 7). doi:10.1161/atvbaha.112.300922
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  Objective-The pleiotropic effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) independent of cholesterol-lowering effects are thought to be mediated through inhibition of the Rho/Rho-kinase pathway. However, we have previously demonstrated that the pleiotropic effects of regular-dose statins are mediated mainly through inhibition of the Rac1 signaling pathway rather than the Rho/Rho-kinase pathway, although the molecular mechanisms of the selective inhibition of the Rac1 signaling pathway by regular-dose statins remain to be elucidated. In this study, we tested our hypothesis that small GTP-binding protein GDP dissociation stimulator (SmgGDS) plays a crucial role in the molecular mechanisms of the Rac1 signaling pathway inhibition by statins in endothelial cells. Approach and Results-In cultured human umbilical venous endothelial cells, statins concentration-dependently increased SmgGDS expression and decreased nuclear Rac1. Statins also enhanced SmgGDS expression in mouse aorta. In control mice, the protective effects of statins against angiotensin II-induced medial thickening of coronary arteries and fibrosis were noted, whereas in SmgGDS-deficient mice, the protective effects of statins were absent. When SmgGDS was knocked down by its small interfering RNA in human umbilical venous endothelial cells, statins were no longer able to induce Rac1 degradation or inhibit angiotensin II-induced production of reactive oxygen species. Finally, in normal healthy volunteers, statins significantly increased SmgGDS expression with a significant negative correlation between SmgGDS expression and oxidative stress markers, whereas no correlation was noted with total or low-density lipoproteincholesterol. Conclusions-These results indicate that statins exert their pleiotropic effects through SmgGDS upregulation with a resultant Rac1 degradation and reduced oxidative stress in animals and humans. © 2013 American Heart Association, Inc.
• Toque, H. A., Nunes, K. P., Yao, L., Liao, J. K., Webb, R. C., Caldwell, R. B., & Caldwell, R. W. (2013). Activated rho kinase mediates diabetes-induced elevation of vascular arginase activation and contributes to impaired corpora cavernosa relaxation: Possible involvement of p38 MAPK activation. Journal of Sexual Medicine, 10(Issue 6). doi:10.1111/jsm.12134
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  Introduction.: Activated RhoA/Rho kinase (ROCK) has been implicated in diabetes-induced erectile dysfunction. Earlier studies have demonstrated involvement of ROCK pathway in the activation of arginase in endothelial cells. However, signaling pathways activated by ROCK in the penis remain unclear. Aim.: We tested whether ROCK and p38 MAPK are involved in the elevation of arginase activity and subsequent impairment of corpora cavernosal (CC) relaxation in diabetes. Methods.: Eight weeks after streptozotocin-induced diabetes, vascular functional studies, arginase activity assay, and protein expression of RhoA, ROCK, phospho-p38 MAPK, p38 MAPK, phospho-MYPT-1Thr850, MYPT-1 and arginase levels were assessed in CC tissues from nondiabetic wild type (WT), diabetic (D) WT (WT+D), partial ROCK 2+/- knockout (KO), and ROCK 2+/- KO+D mice. Main Outcome Measures.: The expression of RhoA, ROCK 1 and 2, phosphorylation of MYPT-1Thr850 and p38 MAPK, arginase activity/expression, endothelial- and nitrergic-dependent relaxation of CC was assayed. Results.: Diabetes significantly reduced maximum relaxation (Emax) to both endothelium-dependent acetylcholine (WT+D: Emax; 61±4% vs. WT: Emax; 75±2%) and nitrergic nerve stimulation. These effects were associated with increased expression of active RhoA, ROCK 2, phospho-MYPT-1Thr850, phospho-p38 MAPK, arginase II, and activity of corporal arginase (1.6-fold) in WT diabetic CC. However, this impairment in CC of WT+D mice was absent in heterozygous ROCK 2+/- KO+D mice for acetylcholine (Emax: 80±5%) and attenuated for nitrergic nerve-induced relaxation. CC of ROCK 2+/- KO+D mice showed much less ROCK activity, did not exhibit p38 MAPK activation, and had reduced arginase activity and arginase II expression. These findings indicate that ROCK 2 mediates diabetes-induced elevation of arginase activity. Additionally, pretreatment of WT diabetic CC with inhibitors of arginase (ABH) or p38 MAPK (SB203580) partially prevented impairment of ACh- and nitrergic nerve-induced relaxation and elevation of arginase activity. Conclusion.: ROCK 2, p38 MAPK and arginase play key roles in diabetes-induced impairment of CC relaxation. © 2013 International Society for Sexual Medicine.
• Dong, M., Liao, J. K., Fang, F., Lee, A. P., Yan, B. P., Liu, M., & Yu, C. M. (2012). Increased Rho kinase activity in congestive heart failure. European Journal of Heart Failure, 14(Issue 9). doi:10.1093/eurjhf/hfs068
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  Aims Rho kinases (ROCKs) are the best characterized effectors of the small G-protein RhoA, and play a role in enhanced vasoconstriction in animal models of congestive heart failure (CHF). This study examined if ROCK activity is increased in CHF and how it is associated with the outcome in CHF.Methods and resultsPatients admitted with CHF (n 178), disease controls (n 31), and normal subjects (n 30) were studied. Baseline ROCK activity was measured by phosphorylation of themyosin-binding subunit in peripheral leucocytes. The patients were followed up for 14.4 ± 7.2 months (range 0.526 months) or until the occurrence of cardiac death. The ROCK activity in CHF patients (2.93 ± 0.87) was significantly higher than that of the disease control (2.06 ± 0.38, P < 0.001) and normal control (1.57 ± 0.43, P < 0.001) groups. Similarly, protein levels of ROCK1 and ROCK2 as well as the activity of RhoA in CHF were significantly higher than in disease controls and normal controls (all P < 0.05). Dyspnoea at rest (β 0.338, P < 0.001), low left ventricular ejection fraction (β 0.277, P < 0.001), and high creatinine (β 0.202, P 0.006) were independent predictors of the baseline ROCK activity in CHF. Forty-five patients died within 2 years follow-up (25.3). Combining ROCK activity and N-terminal pro brain natriuretic peptide (NT-proBNP) had an incremental value (log rank χ2 11.62) in predicting long-term mortality when compared with only NT-proBNP (log rank χ2 5.16, P < 0.05).ConclusionROCK activity is increased in CHF and it might be associated with the mortality in CHF. ROCK activity might be a complementary biomarker to CHF risk stratification. © 2012 The Author.
• Fujimura, N., Noma, K., Hata, T., Soga, J., Hidaka, T., Idei, N., Fujii, Y., Mikami, S., Maruhashi, T., Iwamoto, Y., Kihara, Y., Chayama, K., Kato, H., Liao, J. K., & Higashi, Y. (2012). Mineralocorticoid receptor blocker eplerenone improves endothelial function and inhibits Rho-associated kinase activity in patients with hypertension. Clinical Pharmacology and Therapeutics, 91(Issue 2). doi:10.1038/clpt.2011.227
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  Hypertension is associated with endothelial dysfunction and activated Rho-associated kinases (ROCKs). The purpose of this study was to evaluate the effects of the selective mineralocorticoid receptor blocker, eplerenone, on endothelial function and ROCK activity in patients with hypertension. The study was carried out over 48 weeks in 60 untreated patients with hypertension who were randomly assigned to eplerenone, nifedipine, and losartan groups. We evaluated the effects of each treatment on flow-mediated vasodilation (FMD) and ROCK activity in peripheral leukocytes. Eplerenone increased FMD and decreased leukocyte ROCK activity. Nifedipine decreased ROCK activity but did not alter FMD. Losartan increased FMD but did not alter ROCK activity. Hypotensive effects were similar in the three groups, as was nitroglycerin-induced vasodilation during the follow-up period. There were no significant differences between the groups with respect to other parameters. The study results show that eplerenone improves endothelial function and inhibits ROCK activity in patients with essential hypertension. © 2012 american Society for Clinical Pharmacology and Therapeutics.
• Hung, M. J., Cherng, W. J., Hung, M. Y., Kuo, L. T., Cheng, C. W., Wang, C. H., Yang, N. I., & Liao, J. K. (2012). Increased leukocyte Rho-associated coiled-coil containing protein kinase activity predicts the presence and severity of coronary vasospastic angina. Atherosclerosis, 221(Issue 2). doi:10.1016/j.atherosclerosis.2012.01.001
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  Objective: Although inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) has been shown to prevent coronary vasospastic angina (CVA), direct evidence linking ROCK activity and CVA is lacking. Accordingly, we investigated whether ROCK activity is an independent marker for CVA and is altered after treatment with antispastic medications. Methods and results: We prospectively studied 31 Taiwanese patients who were diagnosed with CVA and 33 control subjects. Subject demographics were recorded, and blood samples were obtained at baseline in all participants and in CVA patients after 3 months of antispastic treatment. Compared with control subjects, leukocyte ROCK activity was greater in CVA patients (136% versus 91%, P
• Medvetz, D. A., Khabibullin, D., Hariharan, V., Ongusaha, P. P., Goncharova, E. A., Schlechter, T., Darling, T. N., Hofmann, I., Krymskaya, V. P., Liao, J. K., Huang, H., & Henske, E. P. (2012). Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion.. PloS one, 7(Issue 11). doi:10.1371/journal.pone.0047842
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  Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd(flox/flox) mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.
• Wang, C. Y., & Liao, J. K. (2012). A mouse model of diet-induced obesity and insulin resistance. Methods in Molecular Biology. doi:10.1007/978-1-61779-430-8_27
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  Obesity is reaching pandemic proportions in Western society. It has resulted in increasing health care burden and decreasing life expectancy. Obesity is a complex, chronic disease, involving decades of pathophysiological changes and adaptation. Therefore, it is difficult ascertain the exact mechanisms for this long-term process in humans. To circumvent some of these issues, several surrogate models are available, including murine genetic loss-of-function mutations, transgenic gain-of-function mutations, polygenic models, and different environmental exposure models. The mouse model of diet-induced obesity has become one of the most important tools for understanding the interplay of high-fat Western diets and the development of obesity. The diet-induced obesity model closely mimics the increasingly availability of the high-fat/high-density foods in modern society over the past two decades, which are main contributors to the obesity trend in human. This model has lead to many discoveries of the important signalings in obesity, such as Akt and mTOR. The chapter describes protocols for diet induced-obesity model in mice and protocols for measuring insulin resistance and sensitivity.
• Wang, Q. M., Stalker, T. J., Gong, Y., Rikitake, Y., Scalia, R., & Liao, J. K. (2012). Inhibition of Rho-kinase attenuates endothelial-leukocyte interaction during ischemia-reperfusion injury. Vascular Medicine (United Kingdom), 17(Issue 6). doi:10.1177/1358863x12459790
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  Resuscitation from hemorrhagic shock induces endothelial dysfunction and activates inflammatory cascades leading to organ damage. Following restoration of blood flow to ischemic vascular beds, leukocyte-endothelium interactions leading to leukocyte infiltration into the vascular wall occur very early due, in part, to the loss of endothelium-derived nitric oxide (NO). The mechanism by which ischemia-reperfusion injury impairs endothelium-derived NO is not completely understood. We hypothesized that inhibition of Rho-kinase could exert beneficial effects following hemorrhagic shock by preserving endothelial function and attenuating leukocyte trafficking in the microcirculation. Using intravital microscopy, we found that resuscitation from hemorrhage acutely increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. Treatment of mice with the Rho-kinase inhibitor fasudil, markedly attenuated leukocyte-endothelium interaction in response to hemorrhage/reinfusion. The beneficial effect of fasudil was not observed in endothelial nitric oxide synthase (eNOS)-/- mice. In conclusion, inhibition of Rho-kinase prevents inflammatory leukocyte trafficking in the microcirculation via an eNOS-dependent mechanism. Our data support a role for Rho-kinase inhibitors in the treatment of ischemia-reperfusion injury. © The Author(s) 2012.
• Zhou, Q., Mei, Y., Shoji, T., Han, X., Kaminski, K., Oh, G. T., Ongusaha, P. P., Zhang, K., Schmitt, H., Moser, M., Bode, C., & Liao, J. K. (2012). Rho-associated coiled-coil-containing kinase 2 deficiency in bone marrow-derived cells leads to increased cholesterol efflux and decreased atherosclerosis. Circulation, 126(Issue 18). doi:10.1161/circulationaha.111.086041
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  Background-Macrophages play a central role in the development of atherosclerosis. However, the signaling pathways that regulate their function are not well understood. The Rho-associated coiled-coil-containing kinases (ROCK1 and ROCK2) are serine-threonine protein kinases that are involved in the regulation of the actin cytoskeleton. Recent studies suggest that ROCK1 in macrophages and bone marrow-derived cells mediates atherogenesis. However, a similar role for ROCK2-/- in the pathogenesis of atherosclerosis has not been determined. Methods and Results-The bone marrows from wild-type, ROCK2-/-, and ROCK2-/- mice were transplanted into irradiated recipient low-density lipoprotein receptor mice, and atherosclerosis was induced with a 16-week high-cholesterol diet. Compared with wild-type bone marrow-transplanted mice, ROCK2-/- bone marrow-transplanted and ROCK2-/- bone marrow-transplanted mice showed substantially less lipid accumulation in the aorta (8.46±1.42% and 9.80±2.34% versus 15.64±1.89%; P
• Hata, T., Goto, C., Soga, J., Hidaka, T., Fujii, Y., Idei, N., Fujimura, N., Maruhashi, T., Mikami, S., Kihara, Y., Chayama, K., Noma, K., Liao, J. K., & Higashi, Y. (2011). Measurement of Rho-associated kinase (ROCK) activity in humans: Validity of leukocyte p-MBS/t-MBS in comparison with vascular response to fasudil. Atherosclerosis, 214(Issue 1). doi:10.1016/j.atherosclerosis.2010.10.005
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  Background: Rho-associated kinases (ROCKs) have been shown to be involved in the pathogenesis of atherosclerosis. It is clinically important to estimate the degree of ROCK activity in humans. The purpose of this study was to confirm the validity of a leukocyte ROCK parameter as an index of ROCK activity in comparison with vascular response to a ROCK inhibitor. Methods and results: We evaluated the ratio of phospho myosin-binding subunit (p-MBS) on myosin light-chain phosphatase to total MBS in peripheral leukocytes by Western blot analysis and forearm blood flow (FBF) response to the ROCK inhibitor fasudil using strain-gauge plethysmography in 36 healthy subjects and 39 patients with cardiovascular diseases. Fasudil (3, 10, 30 μg/min) was infused intra-arterially for 5 min at each dose. Leukocyte p-MBS/total-MBS was higher in cardiovascular diseases than in healthy subjects (0.97 ± 0.37 vs. 0.51 ± 0.14; P= 0.002). Fasudil increased FBF from 4.9 ± 1.2 to 14.5 ± 5.7. mL/min/100 mL tissue (P< 0.0001) in patients with cardiovascular diseases, while fasudil did not alter FBF in healthy subjects. There was a significant relationship between leukocyte p-MBS/total-MBS and maximal FBF response to fasudil in all subjects (r= 0.72, P< 0.0001). There was also a significant correlation between p-MBS/total-MBS and maximal FBF response to fasudil in patients with cardiovascular diseases (r= 0.59, P< 0.0001). In healthy subjects, there was no significant correlation between the two parameters. Conclusions: These findings suggest that assessment of leukocyte ROCK activity is minimally invasive and does not require pharmacologic intervention using ROCK inhibitors. Leukocyte p-MBS/total-MBS may be useful for evaluating ROCK activity in a clinical setting. © 2010 Elsevier Ireland Ltd.
• Hata, T., Soga, J., Hidaka, T., Idei, N., Fujii, Y., Fujimura, N., Mikami, S., Maruhashi, T., Kihara, Y., Chayama, K., Kato, H., Noma, K., Liao, J. K., & Higashi, Y. (2011). Calcium channel blocker and Rho-associated kinase activity in patients with hypertension. Journal of Hypertension, 29(Issue 2). doi:10.1097/hjh.0b013e328340902d
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  Background: Rho-associated kinases (ROCKs) play an important role in Ca sensitization and vascular resistance. Activation of ROCKs is associated with hypertension. The purpose of this study was to evaluate the effect of the calcium channel blocker amlodipine on ROCKs activity in patients with hypertension. Methods: We evaluated ROCK activity in peripheral leukocytes by Western blot analysis in 651 patients with hypertension treated with antihypertensive agents, 28 untreated hypertensive patients and 28 healthy individuals, and the effects of treatment with amlodipine or losartan for 12 weeks on ROCK activity in 28 untreated hypertensive patients who were randomly divided into an amlodipine group (n = 14) and a losartan group (n = 14). ROCK activity was defined as the ratio of phospho myosin-binding subunit (MBS) on myosin light-chain phosphatase to total MBS. Results: Leukocyte ROCK activity was greater in untreated patients with essential hypertension than in the healthy individuals (0.84 ± 0.24 vs. 0.61 ± 0.18, P = 0.03). In 651 patients with hypertension treated with antihypertensive agents, ROCK activity was significantly lower in the calcium channel blocker-treated group than in the groups treated with renin-angiotensin system inhibitors, diuretics, and β-blockers (0.70 ± 0.24 vs. 0.85 ± 0.29, 0.83 ± 0.24, and 0.86 ± 0.31, P < 0.05, respectively). ROCK activity after 4 and 12 weeks of treatment was significantly decreased in the amlodipine group (0 weeks: 0.85 ± 0.25, 4 weeks: 0.66 ± 0.16, 12 weeks: 0.64 ± 0.15, P < 0.05, respectively) but not in the losartan group, whereas the antihypertensive effects were similar in the two groups. Conclusion: These findings suggest that calcium channel blocker amlodipine inhibits ROCK activity in patients with hypertension. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.
• Huh, Y. H., Zhou, Q., Liao, J. K., & Kitazawa, T. (2011). ROCK inhibition prevents fetal serum-induced alteration in structure and function of organ-cultured mesenteric artery. Journal of Muscle Research and Cell Motility, 32(Issue 2). doi:10.1007/s10974-011-9252-y
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  Chronic treatment with fetal bovine serum (FBS) causes contractility reduction, morphological alteration and DNA synthesis in organ-cultured vascular tissues. Here, we tested the hypothesis that chronic inhibition of ROCK has a protective effect on FBS-induced alterations in small arteries. Rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor. Chronic Y-27632 treatment prevented FBS-induced gradual arterial constriction, wall thickening, reduced contractility, and increased ROCK-specific MYPT1 Thr853 phosphorylation. Treatment with Y-27632 also prevented decreased eNOS mRNA expression, and reduced acetylcholine-induced relaxation. Sudden application of Y-27632 to pre-cultured rings reduced MYPT1 phosphorylation and re-widened the constricted rings. Chronic treatment with Y-27632, however, rather augmented than reduced the FBS-induced RhoA over-expression, also increased ROCK1 and MYPT1 expression and averted the FBS-induced reduction of MLC expression, suggesting a compensation of inhibited RhoA/ROCK activity. Sudden removal of Y-27632 caused a rebound in MYPT1 phosphorylation and vasoconstriction in rabbit mesenteric artery. To test which ROCK isoform has greater involvement in FBS-induced contraction, haploinsufficient Rock1 +/- and Rock2 +/- mouse mesenteric arterial rings were subjected to organ-culture. FBS-induced contraction and RhoA over-expression in either heterozygous animal was not different from wild-type animals. These results suggest that FBS-induced contraction is mediated by up-regulation of RhoA and subsequent activation of ROCK. In conclusion, chronic ROCK inhibition produces some effects that protect against FBS-stimulated vasoconstriction and remodeling. There are also negative effects that a sudden withdrawal of ROCK inhibitor might cause a stronger vasoconstriction than before it was used. © 2011 Springer Science+Business Media B.V.
• Kikuchi, R., Takeshita, K., Uchida, Y., Kondo, M., Cheng, X. W., Nakayama, T., Yamamoto, K., Matsushita, T., Liao, J. K., & Murohara, T. (2011). Pitavastatin-induced angiogenesis and arteriogenesis is mediated by Notch1 in a murine hindlimb ischemia model without induction of VEGF. Laboratory Investigation, 91(Issue 5). doi:10.1038/labinvest.2011.5
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  Notch signaling is reported to regulate angiogenesis, interacting with vascular endothelial growth factor (VEGF) signaling. HMG CoA reductase inhibitors (statins) also alter Notch signaling in vascular cells, but the mechanism and involvement of Notch and VEGF signaling in statin-mediated angiogenesis remain unclear. Here, we examined how statins activate the endothelial Notch1, and promote angiogenesis and arteriogenesis. We examined blood flow recovery after hindlimb ischemia in wild-type (WT) and Notch1 mutant mice treated with or without pitavastatin (3 mg/kg/day, p.o.). Although VEGF induction was not altered in ischemic limbs, pitavastatin promoted blood flow recovery in ischemic limbs in control mice but not in Notch1 mutant mice. Furthermore, pitavastatin induced endothelial ephrinB2 downstream of Notch1 and increased the density of both capillaries and arterioles in the ischemic limbs of WT but not of Notch1 mutant mice. Pitavastatin (100 nmol/l) rapidly activated γ-secretase and Notch1 in human umbilical vein endothelial cells without VEGF induction, which was suppressed by pharmacological inhibition and knockdown of Akt. Pitavastatin also augmented endothelial proliferation and tube formation on Matrigel, which were suppressed by either γ-secretase inhibition or knockdown of Notch1. Pitavastatin-induced microvascular sprouting was also impaired in Notch1 mutant aortic explants. Taken together, pitavastatin activates Notch1 through Akt-dependent stimulation of γ-secretase in endothelial cells, and thereby increases vasculogenesis without VEGF induction. © 2011 USCAP, Inc All rights reserved.
• Li, Y., Hiroi, Y., Ngoy, S., Okamoto, R., Noma, K., Wang, C. Y., Wang, H. W., Zhou, Q., Radtke, F., Liao, R., & Liao, J. K. (2011). Notch1 in bone marrow-derived cells mediates cardiac repair after myocardial infarction. Circulation, 123(Issue 8). doi:10.1161/circulationaha.110.947531
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  Background- The signaling mechanisms that regulate the recruitment of bone marrow (BM)-derived cells to the injured heart are not well known. Notch receptors mediate binary cell fate determination and may regulate the function of BM-derived cells. However, it is not known whether Notch1 signaling in BM-derived cells mediates cardiac repair after myocardial injury. Methods and Results- Mice with postnatal cardiac-specific deletion of Notch1 exhibit infarct size and heart function after ischemic injury that is similar to that of control mice. However, mice with global hemizygous deletion of Notch1 (N1) developed larger infarct size and worsening heart function. When the BM of N1 mice were transplanted into wild-type (WT) mice, infarct size and heart function were worsened and neovascularization in the infarct border area was reduced compared with WT mice transplanted with WT BM. In contrast, transplantation of WT BM into N1 mice lessened the myocardial injury observed in N1 mice. Indeed, hemizygous deletion of Notch1 in BM-derived cells leads to decreased recruitment, proliferation, and survival of mesenchymal stem cells (MSC). Compared with WT MSC, injection of N1 MSC into the infarcted heart leads to increased myocardial injury whereas injection of MSC overexpressing Notch intracellular domain leads to decreased infarct size and improved cardiac function. Conclusions- These findings indicate that Notch1 signaling in BM-derived cells is critical for cardiac repair and suggest that strategies that increase Notch1 signaling in BM-derived MSC could have therapeutic benefits in patients with ischemic heart disease. Copyright © 2011 American Heart Association. All rights reserved.
• Reis, S. A., Thompson, M. N., Lee, J. M., Fossale, E., Kim, H. H., Liao, J. K., Moskowitz, M. A., Shaw, S. Y., Dong, L., Haggarty, S. J., MacDonald, M. E., & Seong, I. S. (2011). Striatal neurons expressing full-length mutant huntingtin exhibit decreased N-cadherin and altered neuritogenesis. Human Molecular Genetics, 20(Issue 12). doi:10.1093/hmg/ddr127
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  The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that maycontribute toHDstriatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in HdhQ111 striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdhQ111 striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdhQ111 striatal cells displayed profound deficits in calcium-dependent N-cadherinmediated cell clustering and cell-substratum adhesion, and primary HdhQ111 striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process. © The Author 2011. Published by Oxford University Press. All rights reserved.
• Soga, J., Noma, K., Hata, T., Hidaka, T., Fujii, Y., Idei, N., Fujimura, N., Mikami, S., Maruhashi, T., Kihara, Y., Chayama, K., Kato, H., Liao, J. K., & Higashi, Y. (2011). Rho-associated kinase activity, endothelial function, and cardiovascular risk factors. Arteriosclerosis, Thrombosis, and Vascular Biology, 31(Issue 10). doi:10.1161/atvbaha.111.227892
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  OBJECTIVE-: Cardiovascular diseases are associated with chronic activation of Rho-associated kinases (ROCKs) and endothelial dysfunction. Both increased ROCK activity and endothelial dysfunction are thought to be closely associated with conventional cardiovascular risk factors. The purpose of this study was to determine the relationships between ROCK activity, endothelial function, and cardiovascular risk factors. METHODS AND RESULTS-: We evaluated ROCK activity in peripheral leukocytes by Western blot analysis and flow-mediated vasodilation by ultrasonography in 242 men who had no cardiovascular or cerebrovascular diseases (mean age, 40±10 years; range, 20 to 73 years). ROCK activity was defined as the ratio of phospho myosin-binding subunit on myosin light chain phosphatase to total myosin-binding subunit. Univariate regression analysis revealed that leukocyte ROCK activity significantly correlated with body mass index (r=0.29, P=0.003); systolic blood pressure (r=0.25, P=0.01); low-density lipoprotein cholesterol level (r=0.21, P=0.04); and Framingham risk factor score, a cumulative cardiovascular risk index for heart attack (r=0.31, P
• Wei, Y., Yemisci, M., Kim, H. H., Yung, L. M., Shin, H. K., Hwang, S. K., Guo, S., Qin, T., Alsharif, N., Brinkmann, V., Liao, J. K., Lo, E. H., & Waeber, C. (2011). Fingolimod provides long-term protection in rodent models of cerebral ischemia. Annals of Neurology, 69(Issue 1). doi:10.1002/ana.22186
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  Objective: The sphingosine-1-phosphate (S1P) receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver, and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species. Methods: We used rodent models of middle cerebral artery occlusion and cell-culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod. Results: In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema, and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by tumor necrosis factor alpha. Interpretation: These findings suggest that anti-inflammatory mechanisms, and possibly vasculoprotection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment. © 2011 American Neurological Association.
• Zhu, M., Liu, P. Y., Kasahara, D. I., Williams, A. S., Verbout, N. G., Halayko, A. J., Fedulov, A., Shoji, T., Williams, E. S., Noma, K., Shore, S. A., & Liao, J. K. (2011). Role of Rho kinase isoforms in murine allergic airway responses. European Respiratory Journal, 38(Issue 4). doi:10.1183/09031936.00125010
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  Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Wildtype (WT)mice and heterozygousROCK1 andROCK2 knockoutmice (ROCK1 +/- and ROCK2 +/-, respectively) were sensitised and challenged with ovalbumin. ROCK expression and activation were assessed by western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Compared with WT mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1 +/-and ROCK2 +/- mice, respectively, without changes in the other isoform. In WT lungs, ROCK activation increased after ovalbumin challenge and was sustained for several hours. This activation was reduced in ROCK1 +/- and ROCK2 +/- lungs. Airway responsiveness was comparable in WT, ROCK1 +/-, and ROCK2 +/- mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in WT, but not ROCK1 +/- or ROCK2 +/- mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1 +/- and ROCK2 +/- versus WT mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5 and lymphocytes were also reduced in ROCK1 +/- mice. In conclusion, both ROCK1 and ROCK2 are important in regulating allergic airway responses. Copyright©ERS 2011.
• Akashi, M., Soma, H., Yamamoto, T., Tsugitomi, A., Yamashita, S., Yamamoto, T., Nishida, E., Yasuda, A., Liao, J. K., & Node, K. (2010). Noninvasive method for assessing the human circadian clock using hair follicle cells. Proceedings of the National Academy of Sciences of the United States of America, 107(Issue 35). doi:10.1073/pnas.1003878107
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  A thorough understanding of the circadian clock requires qualitative evaluation of circadian clock gene expression. Thus far, no simple and effective method for detecting human clock gene expression has become available. This limitation has greatly hampered our understanding of human circadian rhythm. Here we report a convenient, reliable, and less invasive method for detecting human clock gene expression using biopsy samples of hair follicle cells from the head or chin. We show that the circadian phase of clock gene expression in hair follicle cells accurately reflects that of individual behavioral rhythms, demonstrating that this strategy is appropriate for evaluating the human peripheral circadian clock. Furthermore, using this method, we indicate that rotating shift workers suffer from a serious time lag between circadian gene expression rhythms and lifestyle. Qualitative evaluation of clock gene expression in hair follicle cells, therefore, may be an effective approach for studying the human circadian clock in the clinical setting.
• Biswas, P. S., Gupta, S., Chang, E., Song, L., Stirzaker, R. A., Liao, J. K., Bhagat, G., & Pernis, A. B. (2010). Phosphorylation of IRF4 by ROCK2 regulates IL-17 and IL-21 production and the development of autoimmunity in mice. Journal of Clinical Investigation, 120(Issue 9). doi:10.1172/jci42856
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  Deregulated production of IL-17 and IL-21 plays a key pathogenic role in many autoimmune disorders. A delineation of the mechanisms that underlie the inappropriate synthesis of IL-17 and IL-21 in autoimmune diseases can thus provide important insights into potential therapies for these disorders. Here we have shown that the serine-threonine kinase Rho-associated, coiled-coil-containing protein kinase 2 (ROCK2) becomes activated in mouse T cells under Th17 skewing conditions and phosphorylates interferon regulatory factor 4 (IRF4), a transcription factor that is absolutely required for the production of IL-17 and IL-21. We furthermore demonstrated that ROCK2-mediated phosphorylation of IRF4 regulated the synthesis of IL-17 and IL-21 and the differentiation of Th17 cells. Whereas CD4+ T cells from WT mice activated ROCK2 physiologically under Th17 conditions, CD4+ T cells from 2 different mouse models of spontaneous autoimmunity aberrantly activated ROCK2 under neutral conditions. Moreover, administration of ROCK inhibitors ameliorated the deregulated production of IL-17 and IL-21 and the inflammatory and autoantibody responses observed in these autoimmune mice. Our findings thus uncover a crucial link among ROCK2, IRF4, and the production of IL-17 and IL-21 and support the idea that selective inhibition of ROCK2 could represent an important therapeutic regimen for the treatment of autoimmune disorders.
• Bryan, B. A., Dennstedt, E., Mitchell, D. C., Walshe, T. E., Noma, K., Loureiro, R., Saint-Geniez, M., Campaigniac, J. P., Liao, J. K., & D'Amore, P. A. (2010). RhoA/ROCK signaling is essential for multiple aspects of VEGF-mediated angiogenesis. FASEB Journal, 24(Issue 9). doi:10.1096/fj.09-145102
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  The small GTPase RhoA and its downstream effectors, ROCK1 and ROCK2, regulate a number of cellular processes, including cell motility, proliferation, survival, and permeability. Pharmacological inhibitors of the Rho pathway reportedly block angiogenesis; however, the molecular details of this inhibition are largely unknown. We demonstrate that vascular endothelial growth factor-A (VEGF) rapidly induces RhoA activation in endothelial cells (ECs). Moreover, the pharmacological inhibition of ROCK1/2 using 10 μM Y-27632 (the IC 50 for this compound in ECs) strongly disrupts vasculogenesis in pluripotent embryonic stem cell cultures, VEGF-mediated regenerative angiogenesis in ex vivo retinal explants, and VEGF-mediated in vitro EC tube formation. Furthermore, using small interfering RNA knockdown and mouse heterozygote knockouts of ROCK1 and ROCK2, we provide data indicating that VEGF-driven angiogenesis is largely mediated through ROCK2. These data demonstrate that Rho/ROCK signaling is an important mediator in a number of angiogenic processes, including EC migration, survival, and cell permeability, and suggest that Rho/ROCK inhibition may prove useful for the treatment of angiogenesis-related disorders. © FASEB.
• Hidaka, T., Hata, T., Soga, J., Fujii, Y., Idei, N., Fujimura, N., Kihara, Y., Noma, K., Liao, J. K., & Higashi, Y. (2010). Increased leukocyte rho kinase (ROCK) activity and endothelial dysfunction in cigarette smokers. Hypertension Research, 33(Issue 4). doi:10.1038/hr.2010.3
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  Rho-associated kinases (ROCKs) have been shown to be involved in the pathogenesis of atherosclerosis. Although smoking is associated with endothelial dysfunction and ROCK inhibitors improve endothelial function in smokers, it is not known whether ROCK activity is increased in smokers and whether this correlates with endothelial dysfunction. The purpose of this study was to evaluate the relationship between ROCK activity and endothelial function in smokers. We evaluated flow-mediated vasodilatation (FMD) using ultrasonography and ROCK activity in peripheral leukocytes using western blot analysis in 14 male smokers (28.1±3.9 years) and 15 healthy male non-smokers (28.3±3.6 years). ROCK activity was defined as the ratio of phospho-myosin-binding subunit (MBS) on myosin light-chain phosphatase to total MBS. FMD was significantly less in smokers than in non-smokers (4.7±3.1 vs. 9.0±3.8%, P=0.005). Nitroglycerine-induced vasodilation was similar in the two groups. ROCK activity was greater in smokers than in non-smokers (0.78±0.27 vs. 0.54±018, P=0.012). The expression of total MBS, ROCK1 and ROCK2 were similar in the two groups. ROCK activity correlated with systolic blood pressure (r=-0.42, P=0.039). Multiple regression analysis revealed that smoking is an independent predictor of ROCK activity. There was a significant correlation between FMD and ROCK activity (r-=0.42, P=0.035). No other variable was correlated with FMD. These findings suggest that ROCK activity is enhanced by smoking and is a predictor of endothelial function. © 2010 The Japanese Society of Hypertension All rights reserved.
• Ii, M., Takeshita, K., Ibusuki, K., Luedemann, C., Wecker, A., Eaton, E., Thorne, T., Asahara, T., Liao, J. K., & Losordo, D. W. (2010). Notch signaling regulates endothelial progenitor cell activity during recovery from arterial injury in hypercholesterolemic mice. Circulation, 121(Issue 9). doi:10.1161/circulationaha.105.553917
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  Background: Little is known about the role of endothelial progenitor cells (EPCs) in atherosclerosis. Accordingly, we performed a series of assessments with hypercholesterolemic (apolipoprotein E-null [ApoE]) and wild-type (WT) mice to evaluate how cholesterol influences reendothelialization, atherosclerosis, and EPC function after arterial injury. Methods and results: Unexpectedly, reendothelialization (assessed by resistance to Evans blue staining) and circulating EPC counts (EPC culture assay) were greater in ApoE mice than in WT mice, and transplantation of ApoE bone marrow in WT mice accelerated endothelial recovery and increased recruitment of bone marrow-derived EPCs to the neoendothelium. Cholesterol concentration-dependently promoted the proliferation (MTS assay) of both ApoE and WT EPCs, and the concentration dependence of EPC adhesion (to vitronectin-, collagen type I-, fibronectin-, and laminin-coated plates), migration (modified Boyden chamber assay), and antiapoptotic (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling stain) activity was biphasic. Cholesterol enhanced the messenger RNA expression (quantitative, real-time reverse-transcription polymerase chain reaction) of vascular endothelial growth factor and inhibited Notch1 messenger RNA expression in both ApoE and WT EPCs, whereas endothelial nitric oxide synthase messenger RNA expression increased in ApoE EPCs and declined in WT EPCs after cholesterol exposure. EPC activity was greater in Notch1 EPCs than in WT EPCs, and transplantation of Notch1 bone marrow accelerated endothelial recovery after arterial injury in WT mice. Conclusion: The results presented here provide novel insights into the role of EPCs during atherosclerosis and suggest that cholesterol and Notch1 may be involved in the regulation of EPC activity. Copyright © 2010 American Heart Association. All rights reserved.
• Liao, J. K. (2010). Rac1 and Connective Tissue Growth Factor. The Missing Link Between Atrial Remodeling and the Pathogenesis of Atrial Fibrillation?. Journal of the American College of Cardiology, 55(Issue 5). doi:10.1016/j.jacc.2009.07.071
• Yan, J., Liao, J. K., & Wang, D. (2010). Elevated homocysteine and C-reactive protein levels independently predict worsening prognosis after stroke in Chinese patients. Journal of Huazhong University of Science and Technology - Medical Science, 30(Issue 5). doi:10.1007/s11596-010-0557-7
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  Increased plasma total homocysteine (tHcy) and high sensitivity C-reactive protein (hsCRP) levels are independent risk factors for cardiovascular disease. However, the predictive value of tHcy in combination with hsCRP in patients with stroke is not known. To determine the relationship between tHcy and hsCRP, we enrolled 291 patients with first-onset stroke (196 ischemic and 95 hemorrhagic). Plasma tHcy and hsCRP levels were measured and subsequent vascular events and deaths were determined over a 5-year period. Using the arbitrary cutoff for tHcy (
• Aoyama, T., Takeshita, K., Kikuchi, R., Yamamoto, K., Cheng, X. W., Liao, J. K., & Murohara, T. (2009). γ-Secretase inhibitor reduces diet-induced atherosclerosis in apolipoprotein E-deficient mice. Biochemical and Biophysical Research Communications, 383(Issue 2). doi:10.1016/j.bbrc.2009.03.154
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  Atherosclerosis is a chronic inflammatory disease resulting from interactions between lipids, macrophages and arterial wall cells. The Notch signaling pathway is involved in the activation of macrophages in atherosclerotic lesions. This study examined whether pharmacological inhibition of Notch signaling using a γ-secretase inhibitor (GSI) can reduce atherosclerotic lesion formation. Notch-related molecules were significantly increased in aortas from apolipoprotein E-deficient (ApoE-/-) mice. In particular, macrophages in the plaques showed strong expression of Notch1 and a downstream transcriptional factor, Hes-1. A GSI (LY411,575, 0.2, and 1.0 mg/kg/day) or vehicle control was then administered to ApoE-/- mice fed Western diet for 8 weeks before measuring the expression of Notch-related molecules. Systemic administration of GSI suppressed Notch signaling in vivo and reduced total plaque areas and fatty streak content in the aortic sinus in a dose-dependent manner without serious adverse effects. The GSI also suppressed the migratory activity of macrophages and reduced the expression of intercellular adhesion molecule-1, resulting in significantly decreased macrophage infiltration in the atherosclerotic plaques. These results provided new insight into the anti-atherogenic properties of GSI in Apo E-/- mice fed Western diet. © 2009 Elsevier Inc. All rights reserved.
• Feske, S. K., Sorond, F. A., Henderson, G. V., Seto, M., Hitomi, A., Kawasaki, K., Sasaki, Y., Asano, T., & Liao, J. K. (2009). Increased leukocyte ROCK activity in patients after acute ischemic stroke. Brain Research, 1257(Issue). doi:10.1016/j.brainres.2008.12.045
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  Background: Rho-kinase (ROCK) is a downstream effector of Rho GTPase that is known to regulate various pathological processes important to the development of ischemic stroke, such as thrombus formation, inflammation, and vasospasm. Inhibition of ROCK leads to decreased infarct size in animal models of ischemic stroke. This study tests the hypothesis that ROCK activity increases during the acute phase of ischemic stroke. Methods: Serial blood samples were drawn from 10 patients with acute ischemic stroke presenting within 24 h of symptomonset and with NIHSS scores ≥4. Sampleswere taken at 24, 48, and 72 h. Leukocyte ROCK activity was determined by immunoblotting leukocyte lysates with antibodies to the phosphorylated formofmyosin-binding subunit (P-MBS) ofmyosin light chain phosphatase (MLCP).MBS and P-MBS contents were normalized to α-tubulin, and ROCK activity was expressed as the ratio of P-MBS to MBS. ROCK activities in these 10 patients were compared to baseline ROCK activities in 10 control subjectswithout acute illness andmatched for sex, age, and number of vascular risk factors using a two-tailed Student's t-test. Results: The mean NIHSS score in patients with stroke was 15.4. ROCK activity was significantly increased at 24 and 48 h in patients after acute ischemic stroke when compared to control values, with peak elevations at 48 h after stroke onset. There was no apparent correlation between ROCK activity and stroke severity based on NIHSS. Conclusions: Leukocyte ROCK activity is increased in patients after acute ischemic stroke with maximal activity occurring about 48 h after stroke onset. These findings suggest that activation of ROCK may play a role in the pathogenesis of ischemic stroke in humans. © 2008 Elsevier B.V. All rights reserved.
• Li, Y., Takeshita, K., Liu, P. Y., Satoh, M., Oyama, N., Mukai, Y., Chin, M. T., Krebs, L., Kotlikoff, M. I., Radtke, F., Gridley, T., & Liao, J. K. (2009). Smooth muscle notch1 mediates neointimal formation after vascular injury. Circulation, 119(Issue 20). doi:10.1161/circulationaha.108.790485
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  BACKGROUND-: Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMCs) may contribute to neointimal formation after vascular injury. METHODS AND RESULTS-: We performed carotid artery ligation in wild-type, control (SMC-specific Cre recombinase transgenic [smCre-Tg]), general Notch1 heterozygous deficient (N1), SMC-specific Notch1 heterozygous deficient (smN1), and general Notch3 homozygous deficient (N3) mice. Compared with wild-type or control mice, N1 and smN1 mice showed a 70% decrease in neointimal formation after carotid artery ligation. However, neointimal formation was similar between wild-type and N3 mice. Indeed, SMCs derived from explanted aortas of either N1- or smN1 mice showed decreased chemotaxis and proliferation and increased apoptosis compared with control or N3 mice. This correlated with decreased staining of proliferating cell nuclear antigen-positive cells and increased staining of cleaved caspase-3 in the intima of N1- or smN1 mice. In SMCs derived from CHF1/Hey2 mice, activation of Notch signaling did not lead to increased SMC proliferation or migration. CONCLUSIONS-: These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation after vascular injury through CHF1/Hey2 and suggest that therapies that target Notch1/CHF1/Hey2 in SMCs may be beneficial in preventing vascular proliferative diseases. © 2009 American Heart Association, Inc.
• Liu, P. Y., Liao, J. K., Liu, Y. W., Lin, L. J., & Chen, J. H. (2009). Response to letters regarding article, "Evidence for statin pleiotropy in humans: Differential effects of statins and ezetimibe on rho-associated coiled-coil containing protein kinase activity, endothelial function, and inflammation". Circulation, 120(Issue 9). doi:10.1161/circulationaha.109.863399
• Liu, P. Y., Liu, Y. W., Lin, L. J., Chen, J. H., & Liao, J. K. (2009). Evidence for statin pleiotropy in humans: Differential effects of statins and ezetimibe on Rho-associated coiled-coil containing protein kinase activity, endothelial function, and inflammation. Circulation, 119(Issue 1). doi:10.1161/circulationaha.108.813311
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  Background-By inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase, statins not only reduce cholesterol biosynthesis but also decrease the formation of isoprenoids, which are important for mediating signaling through the Rho-associated coiled-coil containing protein kinase (ROCK) pathway. Increased ROCK activity has been implicated in endothelial dysfunction and vascular inflammation. We hypothesize that ezetimibe, which inhibits intestinal cholesterol absorption, may not exert similar cholesterol-independent or pleiotropic effects of statins and, when used with a lower dose of statin, have less effect on ROCK activity than a higher dose of statin. Methods and Results-In a prospective, randomized, observer-blinded study, we treated 60 dyslipidemic subjects without cardiovascular disease with simvastatin 40 mg/d, simvastatin/ezetimibe 10/10 mg/d, or placebo tablets for 28 days (n=20 in each arm). We evaluated baseline demographics and lipid levels, ROCK activity, C-reactive protein, and flow-mediated dilation before and after treatment. Compared with the placebo group, both treatment regimens decreased low-density lipoprotein cholesterol by 38% and C-reactive protein by 38% to 40% after 28 days (P
• Nohria, A., Prsic, A., Liu, P. Y., Okamoto, R., Creager, M. A., Selwyn, A., Liao, J. K., & Ganz, P. (2009). Statins inhibit Rho kinase activity in patients with atherosclerosis. Atherosclerosis, 205(Issue 2). doi:10.1016/j.atherosclerosis.2008.12.023
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  Background: In addition to inhibiting cholesterol synthesis, statins (HMG-CoA reductase inhibitors) decrease the formation of isoprenoid intermediates required for the activation of key signaling pathways, including Rho/Rho kinase (ROCK). In experimental settings, statins inhibit ROCK and reverse vascular dysfunctions in atherosclerosis, independent of cholesterol reduction. It is not known whether statins inhibit ROCK activity in humans with atherosclerosis. Methods: We investigated 35 patients with stable atherosclerosis in a randomized, double-blind study comparing treatment with high-dose (80 mg/d) or low-dose (10 mg/d) atorvastatin to placebo for 28 days. Blood samples for leukocyte ROCK activity, fasting lipids, and high-sensitivity C-reactive protein (hs-CRP) were obtained on days 0, 7, 14, and 28 after randomization and change over time with the two statin treatments relative to placebo was examined. Results: Atorvastatin 80 mg/d reduced ROCK activity (p = 0.002 vs. placebo). This decline was rapid and significant within 2 weeks of treatment. The inhibition of ROCK by atorvastatin (80 mg/d) remained significant even after controlling for changes in low-density lipoprotein cholesterol (LDL-C) and triglycerides (p = 0.01). Furthermore, there was no correlation between changes in ROCK activity and changes in LDL-C (r = 0.2, p = 0.25) or triglycerides (r = 0.1, p = 0.55). There was a modest correlation between ROCK inhibition and change in hs-CRP among patients randomized to atorvastatin 80 mg/d (r = 0.6, p = 0.07). Conclusions: These first-in-man findings demonstrate that high-dose atorvastatin rapidly inhibits the pro-atherogenic Rho/ROCK pathway, independent of cholesterol reduction. This inhibition may contribute to the clinical benefits of statins. Rho/ROCK may provide a useful therapeutic target in patients with atherosclerosis. © 2008 Elsevier Ireland Ltd. All rights reserved.
• Rawlings, R., Nohria, A., Liu, P. Y., Donnelly, J., Creager, M. A., Ganz, P., Selwyn, A., & Liao, J. K. (2009). Comparison of Effects of Rosuvastatin (10 mg) Versus Atorvastatin (40 mg) on Rho Kinase Activity in Caucasian Men With a Previous Atherosclerotic Event. American Journal of Cardiology, 103(Issue 4). doi:10.1016/j.amjcard.2008.10.008
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  In addition to inhibiting cholesterol biosynthesis, statins also inhibit the formation of isoprenoid intermediates, which are required for the activation of the Rho/Rho kinase (ROCK) pathway. Increased ROCK activity has been implicated in causing endothelial dysfunction and atherosclerosis. However, it is not known whether statins, at doses used to lower cholesterol levels, inhibit ROCK activity in humans with atherosclerosis. Furthermore, it is not known whether lipophilic and hydrophilic statins differ in their ability to inhibit ROCK activity. Accordingly, we enrolled 30 men with stable atherosclerosis (low-density lipoprotein [LDL] ≥100 mg/dL) in a randomized, double-blind study comparing equivalent LDL-lowering doses of a hydrophilic statin (rosuvastatin 10 mg once a day) with a lipophilic statin (atorvastatin 40 mg once a day) for 28 days. We assessed the change in lipids, ROCK activity, and flow-mediated dilation (FMD) of the brachial artery before and after statin therapy. Both treatment groups exhibited comparable 30% to 32% and 42% to 45% reductions in total and LDL cholesterol, respectively. Only atorvastatin reduced triglycerides, and neither statin altered high-density lipoprotein cholesterol. Whereas both statins inhibited ROCK activity (p
• Sawada, N., Kim, H. H., Moskowitz, M. A., & Liao, J. K. (2009). Rac1 is a critical mediator of endothelium-derived neurotrophic activity. Science Signaling, 2(Issue 61). doi:10.1126/scisignal.2000162
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  The therapeutic potential of neurotrophic factors has been hampered by their inability to achieve adequate tissue penetration. Brain blood vessels, however, could be an alternative target for neuro-salvage therapies by virtue of their close proximity to neurons. Here we show that hemizygous deletion of Rac1 in mouse endothelial cells (ECs) attenuates brain injury and edema after focal cerebral ischemia. Microarray analysis of Rac1+/- ECs revealed enrichment of stress response genes, basement membrane components, and neurotrophic factors that could affect neuronal survival. Consistent with these expression profiles, endothelial Rac1 hemizygosity enhanced antioxidative and endothelial barrier capacities and potentiated paracrine neuroprotective activities through the up-regulation of the neurotrophic factor, artemin. Endothelial Rac1, therefore, could be an important therapeutic target for promoting endothelial barrier integrity and neurotrophic activity.
• Wang, C. Y., Kim, H. H., Hiroi, Y., Sawada, N., Salomone, S., Benjamin, L. E., Walsh, K., Moskowitz, M. A., & Liao, J. K. (2009). Obesity increases vascular senescence and susceptibility to ischemic injury through chronic activation of Akt and mTOR. Science Signaling, 2(Issue 62). doi:10.1126/scisignal.2000143
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  Obesity and age are important risk factors for cardiovascular disease. However, the signaling mechanism linking obesity with age-related vascular senescence is unknown. Here we show that mice fed a high-fat diet show increased vascular senescence and vascular dysfunction compared to mice fed standard chow and are more prone to peripheral and cerebral ischemia. All of these changes involve long-term activation of the protein kinase Akt. In contrast, mice with diet-induced obesity that lack Akt1 are resistant to vascular senescence. Rapamycin treatment of diet-induced obese mice or of transgenic mice with long-term activation of endothelial Akt inhibits activation of mammalian target of rapamycin (mTOR) - rictor complex 2 and Akt, prevents vascular senescence without altering body weight, and reduces the severity of limb necrosis and ischemic stroke. These findings indicate that long-term activation of Akt-mTOR signaling links diet-induced obesity with vascular senescence and cardiovascular disease.
• Arboleda-Velasquez, J. F., Zhou, Z., Hwa, K. S., Louvi, A., Kim, H. H., Savitz, S. I., Liao, J. K., Salomone, S., Ayata, C., Moskowitz, M. A., & Artavanis-Tsakonas, S. (2008). Linking Notch signaling to ischemic stroke. Proceedings of the National Academy of Sciences of the United States of America, 105(Issue 12). doi:10.1073/pnas.0709867105
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  Vascular smooth muscle cells (SMCs) have been implicated in the pathophysiology of stroke, the third most common cause of death and the leading cause of long-term neurological disability in the world. However, there is little insight into the underlying cellular pathways that link SMC function to brain ischemia susceptibility. Using a hitherto uncharacterized knockout mouse model of Notch 3, a Notch signaling receptor paralogue highly expressed in vascular SMCs, we uncover a striking susceptibility to ischemic stroke upon challenge. Cellular and molecular analyses of vascular SMCs derived from these animals associate Notch 3 activity to the expression of specific gene targets, whereas genetic rescue experiments unambiguously link Notch 3 function in vessels to the ischemic phenotype. © 2008 by The National Academy of Sciences of the USA.
• Hiroi, Y., Guo, Z., Li, Y., Beggs, A. H., & Liao, J. K. (2008). Dynamic regulation of endothelial NOS mediated by competitive interaction with α-actinin-4 and calmodulin. FASEB Journal, 22(Issue 5). doi:10.1096/fj.07-9309com
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  Alpha-actinins are critical components of the actin cytoskeleton. Here we show that α-actinins serve another important biological function by binding to and competitively inhibiting calcium-dependent activation of endothelial NOS (eNOS). α-actinin-2 was found to associate with eNOS in a yeast two-hybrid screen. In vascular endothelial cells, which only express α-actinin-1 and -4, α-actinin-4 interacted and colocalized with eNOS. Addition of α-actinin-4 directly inhibited eNOS recombinant protein, and overexpression of α-actinin-4 inhibited eNOS activity in eNOS-transfected COS-7 cells and bovine aortic endothelial cells (BAECs). In contrast, knockdown of α-actinin-4 by siRNA increased eNOS activity in BAECs. The α-actinin-4-binding site on eNOS was mapped to a central region comprising the calmodulin-binding domain, and the eNOS-binding site on α-actinin-4 was mapped to the fourth spectrin-like rod domain, R4. Treatment of endothelial cells with a calcium ionophore, A23187, decreased α-actinin-4-eNOS interaction, leading to translocation of α-actinin-4 from plasma membrane to cytoplasm. Indeed, addition of calmodulin displaced α-actinin-4 binding to eNOS and increased eNOS activity. These findings indicate that eNOS activity in vascular endothelial cells is tonically and dynamically regulated by competitive interaction with α-actinin-4 and calmodulin. © FASEB.
• Kim, H. H., Sawada, N., Soydan, G., Lee, H. S., Zhou, Z., Hwang, S. K., Waeber, C., Moskowitz, M. A., & Liao, J. K. (2008). Additive effects of statin and dipyridamole on cerebral blood flow and stroke protection. Journal of Cerebral Blood Flow and Metabolism, 28(Issue 7). doi:10.1038/jcbfm.2008.24
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  Recent studies suggest that dipyridamole (DP) may exert stroke protective effects beyond platelet inhibition. The purpose of this study is to determine whether statin and DP could enhance stroke protection through nitric oxide (NO)-dependent vascular effects. Mice were pretreated with DP (10 to 60 mg/kg, q 12 h, 3 days) alone or in combination with a statin (simvastatin; 0.1 to 20 mg/kg per day, 14 days) before transient intraluminal middle cerebral artery occlusion. Although simvastatin (1 mg/kg per day, 14 days) increased endothelial NO synthase (eNOS) activity by 25% and DP (30 mg/kg, q12 h, 3 days) increased aortic cGMP levels by 55%, neither statin nor DP alone, at these subtherapeutic doses, increased absolute cerebral blood flow (CBF) or conferred stroke protection. However, the combination of subtherapeutic doses of simvastatin and DP increased CBF by 50%, decreased stroke volume by 54%, and improved neurologic motor deficits, all of which were absent in eNOS-deficient mice. In contrast, treatment with aspirin (10 mg/kg per day, 3 days) did not augment the neuroprotective effects of DP and/or simvastatin. These findings indicate that statin and DP exert additive NO-dependent vascular effects and suggest that the combination of statin and DP has greater benefits in stroke protection than statin alone through vascular protection. © 2008 ISCBFM All rights reserved.
• Liao, J. K. (2008). Fine-tuning the angiogenic response to vascular endothelial growth factor. Circulation Research, 103(Issue 3). doi:10.1161/circresaha.108.181628
• Liao, J. K. (2008). Is statin discontinuation an option in patients who have had a stroke? Commentary. Nature Clinical Practice Neurology, 4(Issue 1). doi:10.1038/ncpneuro0687
• Liu, P. Y., Wang, C. Y., & Liao, J. K. (2008). Evidence for pleiotropic effects of statins in clinical trials. Immunology, Endocrine and Metabolic Agents in Medicinal Chemistry, 8(Issue 2). doi:10.2174/187152208784587890
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  Lipid-lowering agents, such as 3-hydroxy-3-methylglutaryl coenzyme A(HMG CoA) Teductase inhibitors, also known as statins, have been shown to reduce cardiovascular events. However, evidence from recent clinical trials suggests that the beneficial effects of statins may be derived from both lipid lowering and non-lipid lowering, or "pleiotropic" effects. Animal studies suggest that some of these pleiotropic effects of statins are mediated by inhibition of Rho kinase (ROCK). ROCK has been shown important in regulation of vascular tone, cell proliferation, inflammation and oxidative stress. In the cardiovascular system RhoA/ROCK pathway has also been implicated in angiogenesis, atherosclerosis, cerebral and coronary vasospasm, cerebral ischemia, hypertension, myocardial hypertrophy, and neointima formation after vascular injury. Indeed, in clinical studies, elevated ROCK activity is associated with cardiovascular risks and inhibition of ROCK leads to improved endothelial function. Therefore, modulation of the RhoA/ROCK pathway by statins may provide additional benefits beyond their lipid lowering effects, especially among high-risk cardiovascular patients. © 2008 Bentham Science Publishers Ltd.
• Noma, K., Rikitake, Y., Oyama, N., Yan, G., Alcaide, P., Liu, P. Y., Wang, H., Ahl, D., Sawada, N., Okamoto, R., Hiroi, Y., Shimizu, K., Luscinskas, F. W., Sun, J., & Liao, J. K. (2008). ROCK1 mediates leukocyte recruitment and neointima formation following vascular injury. Journal of Clinical Investigation, 118(Issue 5). doi:10.1172/jci29226
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  Although Rho-associated kinase (ROCK) activity has been implicated in cardiovascular diseases, the tissue- and isoform-specific roles of ROCKs in the vascular response to injury are not known. To address the role of ROCKs in this process, we generated haploinsufficient Rock1 (Rock1+/-) and Rock2 (Rock2+/-) mice and performed carotid artery ligations. Following this intervention, we found reduced neointima formation in Rock1+/- mice compared with that of WT or Rock2+/- mice. This correlated with decreased vascular smooth muscle cell proliferation and survival, decreased levels proinflammatory adhesion molecule expression, and reduced leukocyte infiltration. In addition, thioglycollate-induced peritoneal leukocyte recruitment and accumulation were substantially reduced in Rock1+/- mice compared with those of WT and Rock2+/- mice. To determine the role of leukocyte-derived ROCK1 in neointima formation, we performed reciprocal bone marrow transplantation (BMT) in WT and Rock1+/- mice. Rock1 +/- to WT BMT led to reduced neointima formation and leukocyte infiltration following carotid ligation compared with those of WT to WT BMT. In contrast, WT to Rock1+/- BMT resulted in increased neointima formation. These findings indicate that ROCK1 in BM-derived cells mediates neointima formation following vascular injury and suggest that ROCK1 may represent a promising therapeutic target in vascular inflammatory diseases.
• Ongusaha, P. P., Qi, H. H., Raj, L., Kim, Y. B., Aaronson, S. A., Davis, R. J., Shi, Y., Liao, J. K., & Lee, S. W. (2008). Identification of ROCK1 as an upstream activator of the JIP-3 to JNK signaling axis in response to UVB damage. Science Signaling, 1(Issue 47). doi:10.1126/scisignal.1161938
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  Although apoptosis triggered by ultraviolet B (UVB)-mediated activation of the c-Jun N-terminal kinase (JNK) pathway is mediated by both intrinsic and extrinsic pathways, the mechanism of initiation of JNK activation remains obscure. Here, we report the characterization of the JNK-interacting protein 3 (JIP-3) scaffolding protein as an interacting partner of Rho-associated kinase 1 (ROCK1), as determined by tandem affinity protein purification. Upon UVB-induced stress in keratinocytes, ROCK1 was activated, bound to JIP-3, and activated the JNK pathway. Moreover, phosphorylation of JIP-3 by ROCK1 was crucial for the recruitment of JNK. Inhibition of the activity of ROCK1 in keratinocytes resulted in decreased activation of the JNK pathway and thus a reduction in apoptosis. ROCK1+/- mice exhibited decreased UVB-mediated activation of JNK and apoptosis relative to wild-type mice. Our findings present a new molecular mechanism by which ROCK1 functions as a UVB sensor that regulates apoptosis, an important event in the prevention of skin cancer. Copyright 2008 by the American Association for the Advancement of Science; all rights reserved.
• Sawada, N., Salomone, S., Kim, H. H., Kwiatkowski, D. J., & Liao, J. K. (2008). Regulation of endothelial nitric oxide synthase and postnatal angiogenesis by rac1. Circulation Research, 103(Issue 4). doi:10.1161/circresaha.108.178897
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  Diminished bioavailability of nitric oxide is a hallmark of endothelial dysfunction and is associated with a broad spectrum of vascular disorders such as impaired angiogenesis. Because Rac1, a Rho family member, mediates cellular motility and generation of reactive oxygen species, it could be involved in the regulation of endothelial nitric oxide production. However, the pathophysiological consequences of postnatal endothelial Rac1 deletion on endothelial function have not been determined. We generated endothelial-specific Rac1 haploinsufficient mice (EC-Rac1) using Cre-loxP technology. The EC-Rac1 mice have decreased expression and activity of endothelial nitric oxide synthase (eNOS), impaired endothelium-dependent vasorelaxation, and mild hypertension compared with control (Rac1) mice. Hind limb ischemia model and aortic capillary sprouting assay showed that eNOS activity and angiogenesis was impaired in EC-Rac1 mice. Indeed, Rac1 promotes eNOS gene transcription through p21-activated kinase but not NADPH oxidase, increases eNOS mRNA stability, and enhances eNOS activity by promoting endothelial uptake of l-arginine. These findings indicate that endothelial Rac1 is essential for endothelium-dependent vasomotor response and ischemia-induced angiogenesis. These effects of Rac1 on endothelial function are largely due to the upregulation of eNOS through multiple mechanisms that are mediated, in part, by p21-activated kinase. Therapeutic strategies to enhance Rac1 function, therefore, may be important for preventing endothelial dysfunction. © 2008 American Heart Association, Inc.
• Wang, C. Y., Wen, M. S., Wang, H. W., Hsieh, I. C., Li, Y., Liu, P. Y., Lin, F. C., & Liao, J. K. (2008). Increased vascular senescence and impaired endothelial progenitor cell function mediated by mutation of circadian gene Per2. Circulation, 118(Issue 21). doi:10.1161/circulationaha.108.790469
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  Background - Alteration of the circadian rhythm and increased vascular senescence are linked to cardiovascular disease. Per2, a circadian gene, is known to regulate endothelium-dependent vasomotion. However, the mechanism by which Per2 affects endothelial function is unknown. We hypothesize that endothelial dysfunction in Per2 mutant (Per2m/m) mice is mediated in part by increased vascular senescence and impaired endothelial progenitor cell (EPC) function. Methods and Results - Endothelial cells from Per2m/m mice exhibit increased protein kinase Akt signaling, greater senescence, and impaired vascular network formation and proliferation. Indeed, Per2 m/m mice have impaired blood flow recovery and developed autoamputation of the distal limb when subjected to hind-limb ischemia. Furthermore, matrigel implantation into Per2m/m mice resulted in less neovascularization. Because EPCs contribute to angiogenesis, we studied the role of Per2 in these cells using bone marrow transplantation. Basal EPC levels were similar between wild-type and Per2m/m mice. However, compared with wild-type bone marrow transplantation mice, EPC mobilization was impaired in Per2m/m bone marrow transplantation mice in response to ischemia or VEGF stimulation. Bone marrow transplantation or infusion of wild-type EPC restored blood flow recovery and prevented autoamputation in Per2m/m mice. Conclusion - These findings indicate that mutation of Per2 causes Akt-dependent senescence and impairs ischemia-induced revascularization through the alteration of EPC function. © 2008 American Heart Association, Inc.
• Wang, H. W., Liu, P. Y., Oyama, N., Rikitake, Y., Kitamoto, S., Gitlin, J., Liao, J. K., & Boisvert, W. A. (2008). Deficiency of ROCK1 in bone marrow-derived cells protects against atherosclerosis in LDLR-/- mice. FASEB Journal, 22(Issue 10). doi:10.1096/fj.08-108829
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  Rho kinases (ROCKs) are serine-threonine protein kinases that regulate the actin cytoskeleton. Recent studies suggest that ROCKs also play an important role in cardiovascular disease. However, the isoform- and tissue-specific role of ROCKs in mediating this process is unknown. Using homologous recombination, we generated mutant mice harboring alleles with homozygous deletion of ROCK1 (ROCK1-/-). Most ROCK1-/- mice die perinatally. However, a few ROCK1-/- mice survive to adulthood, are phenotypically normal, and have no apparent compensatory changes in ROCK2. Using these ROCK1 -/- mice, we show that ROCK1 in bone marrow-derived macrophages is critical to the development of atherosclerosis, in part, by mediating foam cell formation and macrophage chemotaxis. Lipid accumulation and atherosclerotic lesions were reduced in atherosclerosis-prone LDLR-/- mice, whose bone marrows have been replaced with bone marrows derived from ROCK1 -/- mice. Bone marrow-derived ROCK1-deficient macrophages exhibited impaired chemotaxis to monocyte chemotactic protein-1 and showed reduced ability to take up lipids and to develop into foam cells when exposed to modified low-density lipoprotein. These findings indicate that ROCK1 in bone marrow-derived cells is a critical mediator of atherogenesis and suggest that macrophage ROCK1 may be an important therapeutic target for vascular inflammation and atherosclerosis. © FASEB.
• Yan, G., You, B., Chen, S. P., Liao, J. K., & Sun, J. (2008). Tumor necrosis factor-α downregulates endothelial nitric oxide synthase mRNA stability via translation elongation factor 1-α 1. Circulation Research, 103(Issue 6). doi:10.1161/circresaha.108.173963
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  Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS), whose expression is downregulated by tumor necrosis factor (TNF)-α at the posttranscriptional level. To elucidate the molecular basis of TNF-α-mediated eNOS mRNA instability, eNOS 3′ untranslated region (3′-UTR) binding proteins were purified by RNA affinity chromatography from cytosolic fractions of TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). The formation of 3′-UTR ribonucleoprotein complexes, with molecular weight of 52 and 57 kDa, was increased by TNF-α. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of the 52-kDa protein identified 3 peptides that comprise the peptide sequence of translation elongation factor 1-α 1 (eEF1A1). In HUVECs, TNF-α rapidly increased eEF1A1 expression, which is maximal after 1 hour and persists for up to 48 hours. RNA gel mobility-shift and UV cross-linking assays indicated that recombinant glutathione S-transferase-eEF1A1 fusion protein specifically binds to a UC-rich sequence in the 3′-UTR of eNOS mRNA. In addition, the domain III of eEF1A1 mediates the binding of eNOS 3′-UTR in eEF1A1. Overexpression of eEF1A1 markedly attenuated the expression of eNOS and luciferase gene fused with eNOS 3′-UTR in both COS-7 cells and bovine aortic endothelial cells (BAECs). Furthermore, adenovirus-mediated overexpression of eEF1A1 increased eNOS mRNA instability, whereas knockdown of eEF1A1 substantially attenuated TNF-α-induced destabilization of eNOS mRNA and downregulation of eNOS expression in HUVECs. These results indicate that eEF1A1 is a novel eNOS 3′-UTR binding protein that plays a critical role in mediating TNF-α-induced decrease in eNOS mRNA stability. © 2008 American Heart Association, Inc.
• Blondeau, N., Lai, Y., Tyndall, S., Popolo, M., Topalkara, K., Pru, J. K., Zhang, L., Kim, H., Liao, J. K., Ding, K., & Waeber, C. (2007). Distribution of sphingosine kinase activity and mRNA in rodent brain. Journal of Neurochemistry, 103(Issue 2). doi:10.1111/j.1471-4159.2007.04755.x
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  Sphingosine-1-phosphate (S1P) is a lipid mediator that exerts multiple cellular functions through activation of a subfamily of G-protein-coupled receptors. Although there is evidence that S1P plays a role in the developing and adult CNS, little is known about the ability of brain parenchyma to synthesize this lipid. We have therefore analyzed the brain distribution of the enzymatic activity of the S1P synthesizing enzyme, sphingosine kinase (SPHK) [EC:2.7.1.91], as well as mRNA distribution for one of the two isoforms of this enzyme, sphingosine kinase 2. SPHK activity, measured by the conversion of [3H]sphingosine to [3H]S1P, is highest in cerebellum, followed by cortex and brainstem. Lowest activities were found in striatum and hippocampus. Sensitivity to 0.1% Triton-X suggests that this activity is accounted for by SPHK2. RT-PCR and in situ hybridization studies show that mRNA for this isoform has a distribution similar to that of SPHK activity. In vivo and in vitro ischemia increase SPHK activity and SPHK2 mRNA levels. These results indicate that SPHK2 is the predominant S1P-synthesizing isoform in normal brain parenchyma. Its heterogeneous distribution, in particular laminar distribution in cortex, suggests a neuronal localization and a possible role in cortical and cerebellar functions, in normal as well as ischemic brain. © 2007 The Authors.
• Liao, J. K. (2007). Secondary prevention of stroke and transient ischemic attack: Is more platelet inhibition the answer?. Circulation, 115(Issue 12). doi:10.1161/circulationaha.106.653741
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  BACKGROUND - Recurrent cerebrovascular events constitute an estimated 200 000 of the 700 000 strokes reported annually in the United States, which makes secondary stroke prevention an important goal in the management of disease among patients who have experienced stroke or transient ischemic attack. METHODS AND RESULTS - Various pharmacological approaches have been advocated, but the relative efficacy and safety of these regimens has remained the subject of much debate. The results of recent clinical trials on the use of antiplatelet therapy suggest that patients with a history of stroke or transient ischemic attack may constitute a population distinct from patients with coronary or peripheral vascular disease. This may be caused, in part, by the differing etiologies of stroke and the increased vulnerability of cerebral vessels to bleeding. Indeed, dual antiplatelet therapy, which has been found to be beneficial for the treatment of acute coronary syndromes and percutaneous coronary interventions, does not confer secondary stroke protection. The emerging paradigm is that some level of platelet inhibition is required for secondary stroke protection; a level beyond which increased risk of bleeding arises. CONCLUSION - Because the vast majority of patients with ischemic stroke have recurrent stroke or transient ischemic attack, rather than myocardial infarction, as their next event, antiplatelet therapies for these patients should be administered according to what has been shown to be efficacious for secondary stroke protection rather than for myocardial protection. Combination therapies, which provide optimal platelet inhibition as well as vascular protection, may offer the best strategy for secondary stroke protection. © 2007 American Heart Association, Inc.
• Liu, P. Y., Chen, J. H., Lin, L. J., & Liao, J. K. (2007). Increased Rho Kinase Activity in a Taiwanese Population With Metabolic Syndrome. Journal of the American College of Cardiology, 49(Issue 15). doi:10.1016/j.jacc.2006.12.043
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  Objectives: We sought to determine whether Rho kinase (ROCK) activity is increased in a Taiwanese population with metabolic syndrome (MetS). Background: Recent studies suggest that ROCK may be involved in the pathogenesis of MetS, but clinical studies linking ROCK with MetS are lacking. Methods: We studied 40 Taiwanese subjects (60% men, mean age 55.5 ± 5.6 years) who were diagnosed with MetS with National Cholesterol Educational Program Adult Treatment Panel III criteria and 40 age- and gender-matched control subjects. Subject demographics were recorded, and blood samples were obtained. Results: Compared with control subjects, ROCK activity, as determined by phosphorylation of myosin binding subunit (MBS) in leukocytes, was greater in MetS subjects (mean phospho-MBS/MBS ratio 0.46 vs. 0.35, p = 0.002). A cutoff value for ROCK activity of 0.39 predicted the presence of MetS with specificity and sensitivity rates of 70%. Plasma high-sensitivity C-reactive protein was greater (5.5 mg/l, 95% confidence interval [CI] 3.1 to 7.2 mg/l vs. 2.8 mg/l, 95% CI 1.1 to 3.9 mg/l, p = 0.01) and adiponectin was lower (4.9 μg/ml, 95% CI 3.2 to 6.1 μg/ml vs. 5.9 μg/ml, 95% CI 4.2 to 7.5 μg/ml, p = 0.01) in MetS subjects compared with control subjects, but plasma levels of interleukin-6 and tumor necrosis factor-alpha were not different (p > 0.05 for both). Body mass index, waist circumference, fasting glucose, high-sensitivity C-reactive protein, and triglyceride levels were associated with increased levels of ROCK activity. The risk of increased ROCK activity increased with the number of MetS components (p for trend
• Mukai, Y., Wang, C. Y., Rikitake, Y., & Liao, J. K. (2007). Phosphatidylinositol 3-kinase/protein kinase Akt negatively regulates plasminogen activator inhibitor type 1 expression in vascular endothelial cells. American Journal of Physiology - Heart and Circulatory Physiology, 292(Issue 4). doi:10.1152/ajpheart.00868.2006
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  Plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolytic activity and mediates vascular atherothrombotic disease. Endothelial cells (ECs) synthesize and secrete PAI-1, but the intracellular signaling pathways that regulate PAI-1 expression are not entirely known. We hypothesize that the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt pathway, which regulates endothelial function, could modulate PAI-1 expression in ECs. Cultured bovine aortic and human saphenous vein ECs were stimulated with TNF-α, ANG II, insulin, or serum, and PAI-1 expression was determined by Northern and Western analyses. Inhibition of PI3K with wortmannin or LY-294002 enhanced PAI-1 expression induced by these extracellular stimuli. Similarly, overexpression of a dominant-negative mutant of PI3K or Akt increased TNF-α- and insulin-induced PAI-1 expression. The increase in PAI-1 was due to transcriptional and posttranscriptional mechanisms as PI3K inhibitors increased PAI-1 promoter activity and mRNA stability. The induction of PAI-1 by TNF-α and insulin is mediated, in part, by ERK and p38 MAPK. PI3K inhibitors augmented TNF-α- and insulin-induced phosphorylation of these MAPKs. Simvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, which is known to activate PI3K/Akt, blocks TNF-α- and insulin-induced PAI-1 expression. Treatment with PI3K inhibitors reversed the inhibitor effects of simvastatin on TNF-α- and insulin-induced PAI-1 expression. These findings indicate that the PI3K/Akt pathway acts as a negative regulator of PAI-1 expression in ECs, in part, through the downregulation of MAPK pathways. These results suggest that factors that activate the PI3K/Akt pathway in ECs may have therapeutic benefits for atherothrombotic vascular disease. Copyright © 2007 the American Physiological Society.
• Noma, K., Goto, C., Nishioka, K., Jitsuiki, D., Umemura, T., Ueda, K., Kimura, M., Nakagawa, K., Oshima, T., Chayama, K., Yoshizumi, M., Liao, J. K., & Higashi, Y. (2007). Roles of Rho-Associated Kinase and Oxidative Stress in the Pathogenesis of Aortic Stiffness. Journal of the American College of Cardiology, 49(Issue 6). doi:10.1016/j.jacc.2006.06.082
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  Objectives: The purpose of this study was to determine the relationship between Rho-associated kinase (ROCK) activity and aortic stiffness in humans. Background: Epidemiologic studies have shown that there is a relationship between aortic stiffness and cardiovascular complications. Recent evidence suggests that ROCK plays an important role in the process of atherosclerosis. Methods: We evaluated the forearm blood flow (FBF) response to sodium nitroprusside (SNP), a nitric oxide donor, acetylcholine (ACh), an endothelium-dependent vasodilator, and fasudil, a specific ROCK inhibitor, in 51 healthy male subjects (mean age 45.6 ± 3.0 years). The FBF was measured by using a strain-gauge plethysmography. Carotid-femoral pulse wave velocity (cf-PWV) was measured to assess the aortic stiffness using a pulse wave velocimeter. Results: Intra-arterial infusion of SNP alone, ACh alone, or fasudil alone and after co-infusion of NG-monomethyl-L-arginine (L-NMMA), a nitric-oxide synthase inhibitor, significantly increased FBF in a dose-dependent manner (p < 0.01). Multivariate analysis showed that age and number of pack-years smoked were independent predictors of ROCK activity before or after co-infusion of L-NMMA (p < 0.01) and that age and ROCK activity before or after co-infusion of L-NMMA were independent predictors of cf-PWV (p < 0.01). The concentration of serum malondialdehyde-modified low-density lipoprotein, an index of oxidative stress, was significantly correlated with ROCK activity before and after co-infusion of L-NMMA and cf-PWV (p < 0.01). Conclusions: These findings suggest that aging and accumulating smoking habit, which might induce excessive oxidative stress, are involved in ROCK activity in the vasculature, leading to an increase in aortic stiffness in humans. © 2007 American College of Cardiology Foundation.
• Satoh, M., Matter, C. M., Ogita, H., Takeshita, K., Wang, C. Y., Dorn, G. W., & Liao, J. K. (2007). Inhibition of apoptosis-regulated signaling kinase-1 and prevention of congestive heart failure by estrogen. Circulation, 115(Issue 25). doi:10.1161/circulationaha.106.657981
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  BACKGROUND - Epidemiological studies have shown gender differences in the incidence of congestive heart failure (CHF); however, the role of estrogen in CHF is not known. We hypothesize that estrogen prevents cardiomyocyte apoptosis and the development of CHF. METHODS AND RESULTS - 17β-Estradiol (E2, 0.5 mg/60-day release) or placebo pellet was implanted subcutaneously into male Gαq transgenic (Gq) mice. After 8 weeks, E2 treatment decreased the extent of cardiac hypertrophy and dilation and improved contractility in Gq mice. E2 treatment also attenuated nicotinamide adenine dinucleotide phosphate oxidase activity and superoxide anion production via downregulation of Rac1. This correlated with reduced apoptosis in cardiomyocytes of Gq mice. The antioxidative properties of E2 were also associated with increased expression of thioredoxin (Trx), Trx reductases, and Trx reductase activity in the hearts of Gq mice. Furthermore, the activation of apoptosis signal-regulating kinase 1 and its downstream effectors, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, in the hearts of Gq mice was reduced by long-term E2 treatment. Indeed, E2 (10 nmol/L)-treated cardiomyocytes were much more resistant to angiotensin II-induced apoptosis. These antiapoptotic and cardioprotective effects of E2 were blocked by an estrogen receptor antagonist (ICI 182,780) and by a Trx reductase inhibitor (azelaic acid). CONCLUSIONS - These findings indicate that long-term E2 treatment improves CHF by antioxidative mechanisms that involve the upregulation of Trx and inhibition of Rac1-mediated attenuated nicotinamide adenine dinucleotide phosphate oxidase activity and apoptosis signal-regulating kinase 1 /c-Jun N-terminal kinase/p38 mitogen-activated protein kinase-mediated apoptosis. These results suggest that estrogen may be a useful adjunctive therapy for patients with CHF. © 2007 American Heart Association, Inc.
• Shin, H. K., Salomone, S., Potts, E. M., Lee, S. W., Millican, E., Noma, K., Huang, P. L., Boas, D. A., Liao, J. K., Moskowitz, M. A., & Ayata, C. (2007). Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endothelial mechanisms. Journal of Cerebral Blood Flow and Metabolism, 27(Issue 5). doi:10.1038/sj.jcbfm.9600406
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  Rho-kinase is a serine threonine kinase that increases vasomotor tone via its effects on both endothelium and smooth muscle. Rho-kinase inhibition reduces cerebral infarct size in wild type, but not endothelial nitric oxide synthase deficient (eNOS-/-) mice. The mechanism may be related to Rho-kinase activation under hypoxic/ischemic conditions and impaired vasodilation because of downregulation of eNOS activity. To further implicate Rho-kinase in impaired vascular relaxation during hypoxia/ischemia, we exposed isolated vessels from rat and mouse to 60 mins of hypoxia, and showed that hypoxia reversibly abolished acetylcholine-induced eNOS-dependent relaxation, and that Rho-kinase inhibitor hydroxyfasudil partially preserved this relaxation during hypoxia. We, therefore, hypothesized that if hypoxia-induced Rho-kinase activation acutely impairs vasodilation in ischemic cortex, in vivo, then Rho-kinase inhibitors would acutely augment cerebral blood flow (CBF) as a mechanism by which they reduce infarct size. To test this, we studied the acute cerebral hemodynamic effects of Rho-kinase inhibitors in ischemic core and penumbra during distal middle cerebral artery occlusion (dMCAO) in wild-type and eNOS-/- mice using laser speckle flowmetry. When administered 60 mins before or immediately after dMCAO, Rho-kinase inhibitors hydroxyfasudil and Y-27632 reduced the area of severely ischemic cortex. However, hydroxyfasudil did not reduce the area of CBF deficit in eNOS-/- mice, suggesting that its effect on CBF within the ischemic cortex is primarily endothelium-dependent, and not mediated by its direct vasodilator effect on vascular smooth muscle. Our results suggest that Rho-kinase negatively regulates eNOS activity in acutely ischemic brain, thereby worsening the CBF deficit. Therefore, rapid nontranscriptional upregulation of eNOS activity by small molecule inhibitors of Rho-kinase may be a viable therapeutic approach in acute stroke. © 2007 ISCBFM All rights reserved.
• Takeshita, K., Satoh, M., Ii, M., Silver, M., Limbourg, F. P., Mukai, Y., Rikitake, Y., Radtke, F., Gridley, T., Losordo, D. W., & Liao, J. K. (2007). Critical role of endothelial Notch1 signaling in postnatal angiogenesis. Circulation Research, 100(Issue 1). doi:10.1161/01.res.0000254788.47304.6e
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  Notch receptors are important mediators of cell fate during embryogenesis, but their role in adult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notch receptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we show that blood flow recovery and postnatal neovascularization in response to hindlimb ischemia in haploinsufficient global or endothelial-specific Notch1 mice, but not Notch4 mice, were impaired compared with wild-type mice. The expression of vascular endothelial growth factor (VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice, suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells with VEGF increases presenilin proteolytic processing, γ-secretase activity, Notch1 cleavage, and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treating endothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein kinase Akt or infecting endothelial cells with a dominant-negative Akt mutant. Indeed, inhibition of γ-secretase activity leads to decreased angiogenesis and inhibits VEGF-induced endothelial cell proliferation, migration, and survival. Overexpression of the active Notch1 intercellular domain rescued the inhibitory effects of γ-secretase inhibitors on VEGF-induced angiogenesis. These findings indicate that the phosphatidylinositol 3-kinase/Akt pathway mediates γ-secretase and Notch1 activation by VEGF and that Notch1 is critical for VEGF-induced postnatal angiogenesis. These results suggest that Notch1 may be a novel therapeutic target for improving angiogenic response and blood flow recovery in ischemic limbs. © 2007 American Heart Association, Inc.
• Harari, O. A., Alcaide, P., Ahl, D., Luscinskas, F. W., & Liao, J. K. (2006). Absence of TRAM restricts toll-like receptor 4 signaling in vascular endothelial cells to the MyD88 pathway. Circulation Research, 98(Issue 9). doi:10.1161/01.res.0000220105.85182.28
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  Mammalian cells respond to bacterial lipopolysaccharide (LPS) through a cognate receptor: Toll-like receptor 4 (TLR4). The signaling pathways, which link TLR4 to the proinflammatory transcription factor nuclear factor κB (NF-κB), occur through the intracellular docking proteins MyD88 and Trif. We hypothesize that unlike antigen-presenting cells, vascular endothelial cells (ECs) lack the Trif protein TRAM and are therefore incapable of eliciting Trif-dependent immune responses to LPS. Stimulation of wild-type mice with LPS leads to the activation of NF-κB in ECs and macrophages in vitro and in vivo. In contrast to macrophages, LPS did not activate endothelial NF-κB or NF-κB-dependent genes in MyD88 mice, suggesting the absence of a functional Trif pathway in vascular ECs. Indeed, the Trif-dependent gene cxcl10 was not expressed in ECs after LPS stimulation. This correlated with diminished expression of the Trif accessory TIR protein TRAM in ECs. Overexpression of TRAM cDNA in ECs reconstituted LPS-induced Trif-dependent NF-κB activation and cxcl10 promoter activity. The functional absence of TRAM in vascular ECs restricts TLR4 signaling to MyD88-dependent pathway. This is in contrast to macrophages, which respond to LPS via both Trif- and MyD88-dependent pathways. These findings indicate that vascular ECs do not express the Trif-dependent gene subset. This implies that these genes may be dispensable for the endothelial response to bacterial infection and play no role in the endothelial contribution to the development of atherosclerosis. © 2006 American Heart Association, Inc.
• Hiroi, Y., Kim, H. H., Ying, H., Furuya, F., Huang, Z., Simoncini, T., Noma, K., Ueki, K., Nguyen, N. H., Scanlan, T. S., Moskowitz, M. A., Cheng, S. Y., & Liao, J. K. (2006). Rapid nongenomic actions of thyroid hormone. Proceedings of the National Academy of Sciences of the United States of America, 103(Issue 38). doi:10.1073/pnas.0601600103
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  The binding of thyroid hormone to the thyroid hormone receptor (TR) mediates important physiological effects. However, the transcriptional effects of TR mediated by the thyroid response element (TRE) cannot explain many actions of thyroid hormone. We postulate that TR can initiate rapid, non-TRE-mediated effects in the cardiovascular system through cross-coupling to the phosphatidylinositol 3-kinase (PI3-kinase) protein kinase Akt pathway. In vascular endothelial cells, the predominant TR isoform is TRα1. Treatment of endothelial cells with L-3,5,3′-triiodothyronine (T 3) increased the association of TRα1 with the p85α subunit of PI3-kinase, leading to the phosphorylation and activation of Akt and endothelial nitric oxide synthase (eNOS). The activation of Akt and eNOS by T3 was abolished by the PI3-kinase inhibitors, LY294002 and wortmannin, but not by the transcriptional inhibitor, actinomycin D. To determine the physiological relevance of this PI3-kinase/Akt pathway, we administered T3 to mice undergoing transient focal cerebral ischemia. Compared with vehicle, a single bolus infusion of T3 rapidly increased Akt activity in the brain, decreased mean blood pressure, reduced cerebral infarct volume, and improved neurological deficit score. These neuroprotective effects of T3 were greatly attenuated or absent in eNOS-/- and TRα1-/-β-/- mice and were completely abolished in WT mice pretreated with LY294002 or a T3 antagonist, NH-3. These findings indicate that the activation of PI3-kinase Akt pathways can mediate some of the rapid, non-TRE effects of TR and suggest that the activation of Akt and eNOS contributes to some of the acute vasodilatory and neuroprotective effects of thyroid hormone. © 2006 by The National Academy of Sciences of the USA.
• Liang, K. W., Ting, C. T., Yin, S. C., Chen, Y. T., Lin, S. J., Liao, J. K., & Hsu, S. L. (2006). Berberine suppresses MEK/ERK-dependent Egr-1 signaling pathway and inhibits vascular smooth muscle cell regrowth after in vitro mechanical injury. Biochemical Pharmacology, 71(Issue 6). doi:10.1016/j.bcp.2005.12.028
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  Vascular smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of atherosclerosis and post-angioplasty restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian (Coptis chinensis), and is capable of inhibiting SMC contraction and proliferation, yet the exact mechanism is unknown. We therefore investigated the effect of berberine on SMC growth after mechanic injury in vitro. DNA synthesis and cell proliferation assay were performed to show that berberine inhibited serum-stimulated rat aortic SMC growth in a concentration-dependent manner. Mechanical injury with sterile pipette tip stimulated the regrowth of SMCs. Treatment with berberine prevented the regrowth and migration of SMCs into the denuded trauma zone. Western blot analysis showed that activation of the MEK1/2 (mitogen-activated protein kinase kinase 1/2), extracellular signal-regulated kinase (ERK), and up-regulation of early growth response gene (Egr-1), c-Fos and Cyclin D1 were observed sequentially after mechanic injury in vitro. Semi-quantitative reverse-transcription PCR assay further confirmed the increase of Egr-1, c-Fos, platelet-derived growth factor (PDGF) and Cyclin D1 expression in a transcriptional level. However, berberine significantly attenuated MEK/ERK activation and downstream target (Egr-1, c-Fos, Cyclin D1 and PDGF-A) expression after mechanic injury in vitro. Our study showed that berberine blocked injury-induced SMC regrowth by inactivation of ERK/Egr-1 signaling pathway thereby preventing early signaling induced by injury in vitro. The anti-proliferative properties of berberine may be useful in treating disorders due to inappropriate SMC growth. © 2005 Elsevier Inc. All rights reserved.
• Liao, J. K. (2006). Rho-kinase: A potential link between hypercholesterolemia and abnormal vascular smooth muscle contraction. Circulation Research, 99(Issue 3). doi:10.1161/01.res.0000236798.01988.5d
• Miyamoto, A., Wada, R., Inoue, A., Ishiguro, S., Liao, J. K., & Nishio, A. (2006). Role of angiotensin II receptor subtypes in porcine basilar artery: Functional, radioligand binding, and cell culture studies. Life Sciences, 78(Issue 9). doi:10.1016/j.lfs.2005.06.044
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  We aimed to clarify responsiveness to angiotensin (Ang) II in the porcine basilar artery and the role of Ang II receptor subtypes by functional, radioligand binding, and cell culture studies. Ang II induced more potent contractions in the proximal part than in the distal part of isolated porcine basilar arteries. The contraction induced by Ang II was inhibited by the Ang II type 1 (AT1) receptor antagonist losartan, but the Ang II type 2 (AT2) receptor antagonist PD123319 enhanced it. After removal of the endothelium, the effect of losartan remained but the effect of PD123319 was abolished. The specific binding site of [3H]Ang II on the smooth muscle membrane was inhibited by losartan, but not by PD123319. Stimulation of angiotensin II increased nitric oxide (NO) production in cultured basilar arterial endothelial cells. This production was inhibited by PD123319 and the NO synthase inhibitor L-NG-nitroarginine. These results suggest that the contraction induced by Ang II might be mediated via the activation of AT1 receptors on the basilar arterial smooth muscle cells and be modulated via the activation of AT2 receptors on the endothelial cells, followed by NO production. © 2005 Elsevier Inc. All rights reserved.
• Mukai, Y., Rikitake, Y., Shiojima, I., Wolfrum, S., Satoh, M., Takeshita, K., Hiroi, Y., Salomone, S., Kim, H. H., Benjamin, L. E., Walsh, K., & Liao, J. K. (2006). Decreased vascular lesion formation in mice with inducible endothelial-specific expression of protein kinase Akt. Journal of Clinical Investigation, 116(Issue 2). doi:10.1172/jci26223
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  To determine whether endothelial Akt could affect vascular lesion formation, mutant mice with a constitutively active Akt transgene, which could be inducibly targeted to the vascular endothelium using the tet-off system (EC-Akt Tg mice), were generated. After withdrawal of doxycycline, EC-Akt Tg mice demonstrated increased endothelial-specific Akt activity and NO production. After blood flow cessation caused by carotid artery ligation, neointimal formation was attenuated in induced EC-Akt Tg mice compared with noninduced EC-Akt Tg mice and control littermates. To determine the role of eNOS in mediating these effects, mice were treated with Nω-nitro-L- arginine methyl ester (L-NAME). Neointimal formation was attenuated to a lesser extent in induced EC-Akt Tg mice treated with L-NAME, suggesting that some of the vascular protective effects were NO independent. Indeed, endothelial activation of Akt resulted in less EC apoptosis in ligated arteries. Immunostaining demonstrated decreased inflammatory and proliferative changes in induced EC-Akt Tg mice after vascular injury. These findings indicate that endothelial activation of Akt suppresses lesion formation via increased NO production, preservation of functional endothelial layer, and suppression of inflammatory and proliferative changes in the vascular wall. These results suggest that enhancing endothelial Akt activity alone could have therapeutic benefits after vascular injury.
• Nohria, A., Grunert, M. E., Rikitake, Y., Noma, K., Prsic, A., Ganz, P., Liao, J. K., & Creager, M. A. (2006). Rho kinase inhibition improves endothelial function in human subjects with coronary artery disease. Circulation Research, 99(Issue 12). doi:10.1161/01.res.0000251668.39526.c7
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  Investigations from basic biology suggest that activation of the Rho/Rho kinase pathway reduces the bioavailability of nitric oxide (NO) and thereby promotes atherosclerosis and its clinical complications. Yet, little information is available about the relationship of the Rho/Rho kinase pathway to NO bioavailability in humans with atherosclerosis. Accordingly, we determined whether inhibition of Rho kinase augments NO bioavailability and improves endothelial function in human subjects with coronary artery disease (CAD). Thirteen CAD subjects and 16 age- and sex-matched healthy controls were randomly assigned to receive the Rho kinase inhibitor, fasudil, or placebo for 1 month each in a double-blind crossover trial. Flow-mediated, endothelium-dependent and nitroglycerin-induced, endothelium-independent vasodilation were assessed by brachial artery ultrasonography. Rho kinase activity was measured in peripheral leukocytes. Fasudil increased endothelium-dependent vasodilation in CAD subjects from 9.4±1.9% to 13.4±1.9% (P=0.001) but not in healthy controls (from 11.3±1.4% to 7.7±1.1%; P=0.07). Endothelium-independent vasodilation was not affected by fasudil in either CAD or healthy subjects. Fasudil reduced Rho kinase activity by 59±18% in CAD subjects (P=0.001) but not in healthy subjects (by 3±6%; P=0.60). The change in endothelium-dependent vasodilation achieved with fasudil relative to placebo was inversely proportional to Rho kinase inhibition (ie, greater Rho kinase inhibition was associated with larger improvement in endothelium-dependent vasodilation) (r=-0.48; P=0.01). These findings suggest that Rho/Rho kinase activation promotes endothelial dysfunction in humans with atherosclerosis. Inhibition of the Rho/Rho kinase pathway should provide a useful strategy to restore NO bioavailability in humans with atherosclerosis. © 2006 American Heart Association, Inc.
• Satoh, M., Ogita, H., Takeshita, K., Mukai, Y., Kwiatkowski, D. J., & Liao, J. K. (2006). Requirement of Rac1 in the development of cardiac hypertrophy. Proceedings of the National Academy of Sciences of the United States of America, 103(Issue 19). doi:10.1073/pnas.0510444103
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  The development of cardiac hypertrophy is mediated, in part, by increase in NADPH oxidase activity and myocardial oxidative stress. The Rhp GTPase, Rac, regulates NADPH oxidase activity through interaction with gp91phox and p67phox (in which "phox" is phagocyte oxidase). However, it is not known which Rac isoform mediates this effect in the heart. Here we show that Rac1 is critical for generating oxidative stress and producing cardiac hypertrophy in the adult heart. The Rad gene was temporally and specifically deleted in adult mouse cardiomyocytes (c-Rac1-/-). Compared with wild-type or Rac1 heterozygous mice, the hearts of c-Rac1 -/- mice showed decreased gp91phox and p67phox interaction, NADPH oxidase activity, and myocardial oxidative stress in response to angiotensin II (400 ng/kg per day for 2 weeks) stimulation. This result correlated with decreased myocardial hypertrophy. These results indicate that Rac1 is critical for the hypertrophic response in the heart and suggest that therapies which target myocardial Rad may be beneficial in the treatment of cardiac hypertrophy. © 2006 by The National Academy of Sciences of the USA.
• Sun, J., Yan, G., Ren, A., You, B., & Liao, J. K. (2006). FHL2/SLIM3 decreases cardiomyocyte survival by inhibitory interaction with sphingosine kinase-1. Circulation Research, 99(Issue 5). doi:10.1161/01.res.0000239410.65551.b3
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  Sphingosine kinase-1 (SK1) is a key enzyme catalyzing the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). Recent studies suggest that SK1, and its product S1P, regulate diverse biological functions, including cell growth, differentiation, proliferation, and apoptosis. S1P may also play an important role in cardiac development and ischemic preconditioning, but the mechanism underlying these effects is not known. Using a yeast 2-hybrid screen with SK1 as bait and a cardiac cDNA library to identify novel proteins involved in regulating SK1 activity in cardiomyocytes, we identified the LIM-only factor FHL2 (SLIM3) as a SK1-interacting protein in both yeast and mammalian cells. FHL2, but not FHL1 or FHL3, interacted with SK1, and FHL2 colocalized with SK1 in the cytoplasm. The interaction sites with SK1 consisted of at least 4 LIM domains in FHL2, whereas the C-terminal portion of SK1 mediates the binding of FHL2 in SK1. Overexpression of FHL2 attenuated the activity and antiapoptotic effects of SK1. Indeed, endothelin-1, which is a potent survival factor in cardiomyocytes, inhibited FHL2-SK1 association and increased SK1 activity. These findings indicate that FHL2 is a novel inhibitor of SK1 activity in cardiomyocytes and suggest that targeting FHL2 for inhibition may prevent myocardial apoptosis through activation of SK1. © 2006 American Heart Association, Inc.
• Xiang, F., Sakata, Y., Cui, L., Youngblood, J. M., Nakagami, H., Liao, J. K., Liao, R., & Chin, M. T. (2006). Transcription factor CHF1/Hey2 suppresses cardiac hypertrophy through an inhibitory interaction with GATA4. American Journal of Physiology - Heart and Circulatory Physiology, 290(Issue 5). doi:10.1152/ajpheart.01106.2005
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  Pathological cardiac hypertrophy is considered a precursor to clinical heart failure. Understanding the transcriptional regulators that suppress the hypertrophic response may have profound implications for the treatment of heart disease. We report the generation of transgenic mice that overexpress the transcription factor CHF1/Hey2 in the myocardium. In response to the α-adrenergic agonist phenylephrine, they show marked attenuation in the hypertrophic response compared with wild-type controls, even though blood pressure is similar in both groups. Isolated myocytes from transgenic mice demonstrate a similar resistance to phenylephrine-induced hypertrophy in vitro, providing further evidence that the protective effect of CHF1/Hey2 is mediated at the myocyte level. Induction of the hypertrophy marker genes ANF, BNP, and β-MHC in the transgenic cells is concurrently suppressed in vivo and in vitro, demonstrating that the induction of hypertrophy-associated genes is repressed by CHF1/Hey2. Transfection of CHF1/Hey2 into neonatal cardiomyocytes suppresses activation of an ANF reporter plasmid by the transcription factor GATA4, which has previously been shown to activate a hypertrophic transcriptional program. Furthermore, CHF1/Hey2 binds GATA4 directly in coimmunoprecipitation assays and inhibits the binding of GATA4 to its recognition sequence within the ANF promoter. Our findings demonstrate that CHF1/Hey2 functions as an antihypertrophic gene, possibly through inhibition of a GATA4-dependent hypertrophic program. Copyright © 2006 the American Physiological Society.
• Asahi, M., Huang, Z., Thomas, S., Yoshimura, S. I., Sumii, T., Mori, T., Qiu, J., Amin-Hanjani, S., Huang, P. L., Liao, J. K., Lo, E. H., & Moskowitz, M. A. (2005). Protective effects of statins involving both eNOS and tPA in focal cerebral ischemia. Journal of Cerebral Blood Flow and Metabolism, 25(Issue 6). doi:10.1038/sj.jcbfm.9600070
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  Previous studies have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) protect the brain against ischemic injury by upregulating endothelial nitric oxide synthase (eNOS). Here, we tested the hypothesis that statins provide additional beneficial effects by also upregulating endogenous tissue plasminogen activator (tPA) and enhancing clot lysis in a mouse model of embolic focal ischemia. Heterologous blood clots (0.2 mm) were injected into the distal internal carotid artery to occlude blood flow in the middle cerebral artery territory after long-term (14 days) simvastatin, atorvastatin or vehicle treatment. Ischemic lesion volume, neurologic deficits, as well as residual blood clots were measured at 22 h. Reverse transcription-polymerase chain reaction assessed mRNA levels of eNOS, tPA, and the endogenous plasminogen activator inhibitor PAI-1. Ischemic lesion volumes and neurologic deficits were significantly reduced in wild-type mice by both simvastatin and atorvastatin. Statins increased eNOS and tPA mRNA levels but did not change mRNA levels of PAI-1. In eNOS knockout mice, atorvastatin reduced the volume of ischemic tissue and improved neurologic outcomes after arterial occlusion by blood clot emboli. In contrast, statins did not have protective effects in tPA knockout mice after embolic focal ischemia, but only in a filament model where focal ischemia was achieved via mechanical occlusion. These results suggest that statins protect against stroke by multiple mechanisms involving both eNOS and tPA. The involvement of each pathway may be revealed depending on the choice of experimental stroke model. © 2005 ISCBFM All rights reserved.
• Kim, D. K., Kan, D., Veres, T., Normadin, F., Liao, J. K., Kim, H. H., Lee, S. H., Zahn, M., & Muhammed, M. (2005). Monodispersed Fe-Pt nanoparticles for biomedical applications. Journal of Applied Physics, 97(Issue 10). doi:10.1063/1.1860851
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  Monodispersed Fe-Pt nanoparticles are promising candidates for biomedical applications with an average particle diameter of 6.9 nm Fe48 Pt52, 3.3 nm Fe52 Pt48, and 4.2 nm Fe70 Pt30. They are prepared by simultaneous chemical reduction of Pt (acac)2 and thermal decomposition of Fe (CO)5 in the presence of surfactant as an anti-oxidation reagent and amine as a stabilizer. The blocking temperatures, Tb, of 9 K for Fe70 Pt30, 11 K for Fe52 Pt48 and 14.4 K for Fe48 Pt52 and the mean diameter of the spherical magnetic particles were estimated from the calculated volume to be 3.6, 3.1, and 3.8 nm. The cytotoxicity of unmodified Fe-Pt nanoparticles was performed in brain endothelial cells. Fe48 Pt52 nanoparticles were not found to have any significant toxicity on bEnd3 cells during a 24 h period. © 2005 American Institute of Physics.
• Liao, J. K. (2005). Effects of statins on 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibition beyond low-density lipoprotein cholesterol. American Journal of Cardiology, 96(Issue 5). doi:10.1016/j.amjcard.2005.06.009
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  Statins are potent inhibitors of cholesterol biosynthesis and exert beneficial effects in the primary and secondary prevention of coronary artery disease. However, the overall benefits observed with statins appear to occur much earlier and to be greater than what might be expected from changes in lipid levels alone, suggesting effects beyond cholesterol lowering. Indeed, recent studies indicate that some of the cholesterol-independent or "pleiotropic" effects of statins involve improving endothelial function, enhancing the stability of atherosclerotic plaques, decreasing oxidative stress and inflammation, and inhibiting the thrombogenic response. Many of these pleiotropic effects are mediated by inhibition of isoprenoids, which serve as lipid attachments for intracellular signaling molecules. In particular, inhibition of the small guanosine triphosphate-binding proteins Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation, may play an important role in mediating the pleiotropic effects of statins. © 2005 Elsevier Inc. All rights reserved.
• Limbourg, F. P., Takeshita, K., Radtke, F., Bronson, R. T., Chin, M. T., & Liao, J. K. (2005). Essential role of endothelial Notch1 in angiogenesis. Circulation, 111(Issue 14). doi:10.1161/01.cir.0000160870.93058.dd
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  Background - Notch signaling influences binary cell fate decisions in a variety of tissues. The Notch1 receptor is widely expressed during embryogenesis and is essential for embryonic development. Loss of global Notch1 function results in early embryonic lethality, but the cell type responsible for this defect is not known. Here, we identify the endothelium as the primary target tissue affected by Notch1 signaling. Methods and Results - We generated an endothelium-specific deletion of Notch1 using Tie2Cre and conditional Notch1flox/flox mice. Mutant embryos lacking endothelial Notch1 died at approximately embryonic day 10.5 with profound vascular defects in placenta, yolk sac, and embryo proper, whereas heterozygous deletion had no effect. In yolk sacs of mutant embryos, endothelial cells formed a primary vascular plexus indicative of intact vasculogenesis but failed to induce the secondary vascular remodeling required to form a mature network of well-organized large and small blood vessels, which demonstrates a defect in angiogenesis. These vascular defects were also evident in the placenta, where blood vessels failed to invade the placental labyrinth, and in the embryo proper, where defective blood vessel maturation led to pericardial and intersomitic hemorrhage. Enhanced activation of caspase-3 was detected in endothelial and neural cells of mutant mice, which resulted in enhanced apoptotic degeneration of somites and the neural tube. Conclusions - These findings recapitulate the vascular phenotype of global Notch1-/- mutants and indicate an essential cell-autonomous role of Notch1 signaling in the endothelium during vascular development. These results may have important clinical implications with regard to Notch1 signaling in adult angiogenesis. © 2005 American Heart Association, Inc.
• Rikitake, Y., & Liao, J. K. (2005). Rho-kinase mediates hyperglycemia-induced plasminogen activator inhibitor-1 expression in vascular endothelial cells. Circulation, 111(Issue 24). doi:10.1161/circulationaha.105.534024
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  Background - Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with myocardial infarction and stroke, especially in patients with diabetes. The induction of PAI-1 expression by hyperglycemia involves oxidative stress and protein kinase C (PKC). However, the mechanism by which hyperglycemia increases PAI-1 expression is unknown. Methods and Results - Compared with normoglycemia, exposure of human endothelial cells to hyperglycemia, but not mannitol, increased Rho-kinase activity in a time- and concentration-dependent manner. This increase was inhibited by a PKC inhibitor, GF109203X, and antioxidants N-acetylcysteine (NAC) and reduced form of glutathione (GSH). This correlated with inhibition of hyperglycemia-induced PAI-1 expression by GF109203X, NAC, and GSH. Hyperglycemia-increased PAI-1 mRNA and protein levels were inhibited by Rho-kinase inhibitors hydroxyfasudil and Y27632 and by a dominant-negative mutant of Rho-kinase. The mechanism for this inhibition occurs at the level of gene transcription because Rho-kinase inhibitors repress hyperglycemia-stimulated PAI-1 promoter activity without affecting mRNA stability. Hyperglycemia failed to stimulate Rho-kinase activity and PAI-1 expression in heterozygous ROCK I-knockout murine endothelial cells. Conclusions - Hyperglycemia stimulates Rho-kinase activity via PKC- and oxidative stress-dependent pathways, leading to increased PAI-1 gene transcription. These results suggest that inhibition of ROCK I may be a novel therapeutic target for preventing thromboembolic complications of diabetes and cardiovascular disease. © 2005 American Heart Association, Inc.
• Rikitake, Y., Kim, H. H., Huang, Z., Seto, M., Yano, K., Asano, T., Moskowitz, M. A., & Liao, J. K. (2005). Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke, 36(Issue 10). doi:10.1161/01.str.0000181077.84981.11
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  Background and Purpose - Endothelium-derived nitric oxide (NO) plays a pivotal role in vascular protection. The Rho kinase (ROCK) inhibitor, hydroxyfasudil, prevents the downregulation of endothelial NO synthase (eNOS) under hypoxic conditions. However, it is unknown whether inhibition of ROCK can attenuate ischemia-induced endothelial dysfunction and tissue damage in vivo. Methods - Human vascular endothelial cells were treated with increasing concentrations of hydroxyfasudil (0.1 to 100 μmol/L) and eNOS expression and activity were measured. To determine the physiological relevance of eNOS regulation by ROCK, we administered fasudil, which is metabolized to hydroxyfasudil in vivo, to mice for 2 days before subjecting them to middle cerebral artery occlusion. Cerebral blood flow, cerebral infarct size, and neurologic deficit were measured. Results - In a concentration-dependent manner, hydroxyfasudil increased eNOS mRNA and protein expression, resulting in a 1.9- and 1.6-fold increase, respectively, at 10 μmol/L (P
• Rikitake, Y., Oyama, N., Wang, C. Y., Noma, K., Satoh, M., Kim, H. H., & Liao, J. K. (2005). Decreased perivascular fibrosis but not cardiac hypertrophy in ROCK1 +/- haploinsufficient mice. Circulation, 112(Issue 19). doi:10.1161/circulationaha.105.584623
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  Background - Rho GTPase and its downstream target, Rho-associated kinase (ROCK), have been implicated in diverse cardiovascular diseases such as cardiac hypertrophy. However, pharmacological inhibitors of ROCK are not entirely specific, nor can they discriminate between the ROCK isoforms ROCK1 and ROCK2. To determine the specific role of ROCK1 in the development of cardiac hypertrophy, we generated ROCK1+/- haploinsufficient mice and determined whether cardiac hypertrophy and remodeling are decreased in these mice. Methods and Results - Litters of ROCK1-/- mice on C57B1/6 background were markedly underrepresented, suggesting lethality in utero or postnatally. ROCK1+/- mice, however, are viable and fertile with no obvious phenotypic abnormalities. Basal blood pressure, heart rate, and cardiac dimension and function in ROCK1+/- mice were similar to those in wild-type (WT) littermates. Infusion of angiotensin II (400 ng · kg -1 · min-1 for 28 days) or treatment with N G-nitro-L-arginine methyl ester (1 mg/mL in drinking water for 28 days) caused similar increases in systolic blood pressure, left ventricular wall thickness, left ventricular mass, ratio of heart weight to tibial length, and cardiomyocyte size in ROCK1+/- mice and WT littermates. In contrast, perivascular fibrosis in hearts was increased to a lesser extent in ROCK1 +/- mice compared with WT littermates. This was associated with decreased expression of transforming growth factor-β, connective tissue growth factor, and type III collagen. In addition, perivascular fibrosis induced by transaortic constriction or myocardial infarction was decreased in ROCK1+/- mice compared with WT littermates. Conclusions - These findings indicate ROCK1 is critical for the development of cardiac fibrosis, but not hypertrophy, in response to various pathological conditions and suggest that signaling pathways leading to the hypertrophic and profibrotic response of the heart are distinct. © 2005 American Heart Association, Inc.
• Abbruzzese, T. A., Havens, J., Belkin, M., Donaldson, M. C., Whittemore, A. D., Liao, J. K., & Conte, M. S. (2004). Statin therapy is associated with improved patency of autogenous infrainguinal bypass grafts. Journal of Vascular Surgery, 39(Issue 6). doi:10.1016/j.jvs.2003.12.027
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  Objective HMG-CoA reductase inhibitors (statins) broadly reduce cardiovascular events, effects that are only partly related to cholesterol lowering. Recent studies suggest important anti-inflammatory and antiproliferative properties of these drugs. The purpose of this study was to determine the influence of statin therapy on graft patency after autogenous infrainguinal arterial reconstructions. Methods A retrospective analysis of consecutive patients (1999-2001) who underwent primary autogenous infrainguinal reconstructions with a single segment of greater saphenous vein was performed. Patients were categorized according to concurrent use of a statin. Graft lesions (identified by duplex surveillance) and interventions were tabulated. Comparisons between groups were made by using the Fisher exact test for categorical variables and the Student t test for continuous variables. Patency, limb salvage, and survival were compared by log rank test. A stepwise Cox proportional hazards analysis was then employed to ascertain the relative importance of factors influencing graft patency. Results A total of 172 patients underwent 189 primary autogenous infrainguinal arterial reconstructions (94 statin, 95 control) during the study period. The groups were well matched for age, indication, and atherosclerotic risk factors. Procedures were performed primarily for limb salvage (92%), with 65% to an infrapopliteal target. Perioperative mortality (2.6%) and major morbidity (3.2%) were not different between groups. There was no difference in primary patency (74% ± 5% vs 69% ± 6%; P = .25), limb salvage (92% ± 3% vs 90% ± 4%; P = .37), or survival (69% ± 5% vs 63% ± 5%; P = .20) at 2 years. However, patients on statins had higher primary-revised (94% ± 2% vs 83% ± 5%; P < .02) and secondary (97% ± 2% vs 87% ± 4%; P < .02) graft patency rates at 2 years. Of all factors studied by univariate analysis, only statin use was associated with improved secondary patency (P = .03) at 2 years. This was confirmed by multivariate analysis. The risk of graft failure was 3.2-fold higher (95% confidence interval, 1.04-10.04) for the control group. Perioperative cholesterol levels (available in 47% of patients) were not statistically different between groups. Conclusions Statin therapy is associated with improved graft patency after infrainguinal bypass grafting with saphenous vein.
• Abraham, S. S., Osorio, J. C., Homma, S., Wang, J., Thaker, H. M., Liao, J. K., & Mital, S. (2004). Simvastatin Preserves Cardiac Function in Genetically Determined Cardiomyopathy. Journal of Cardiovascular Pharmacology, 43(Issue 3). doi:10.1097/00005344-200403000-00018
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  Endothelial dysfunction characterizes heart failure (HF). Simvastatin (Sim) increases endothelial nitric oxide (NO) independent of lipid-lowering. We evaluated the effect of Sim on cardiac function, apoptosis, and NO availability in HF. Five-month-old cardiomyopathic (CM) hamsters were divided into 2 groups: Sim (20 mg/kg, 6 weeks, n = 6) and Untreated (n = 6). Age-matched normal hamsters served as controls (n = 6). Serial echocardiograms were performed to measure LV function. Myocardial apoptosis, eNOS, and capillary density were measured at 6 weeks. Cardiomyopathic hamsters had lower LV shortening fraction (SF) compared with controls (17% vs 59 ± 2%), higher LV end-diastolic volume (30 ± 3 vs 6 ± 2 mL/m2), and lower LV mass/volume ratio (0.5 ± 0.04 vs 0.72 ± 0.02 mg/ml, P < 0.001). During follow-up, SF decreased (9 ± 2%) and LV volume increased (38 ± 1 mL/m2) in untreated hamsters (P < 0.05 from baseline) but did not change significantly in the Sim group (P < 0.05 vs untreated). Myocardial caspase-3 activity was higher and apoptotic nuclear density was lower in Sim compared with untreated CM hamsters (0.072 ± 0.02% vs 0.107 ± 0.03%, P < 0.01). Myocardial capillary density was highest in the Sim group (P < 0.05). eNOS expression was not different between groups. Sim retards the progression of HF in CM hamsters. This may be related to an increase in coronary microvasculature, increase in NO availability, and decreased apoptosis.
• Beckman, J. A., Liao, J. K., Hurley, S., Garrett, L. A., Chui, D., Mitra, D., & Creager, M. A. (2004). Atorvastatin restores endothelial function in normocholesterolemic smokers independent of changes in low-density lipoprotein. Circulation Research, 95(Issue 2). doi:10.1161/01.res.0000134628.96682.9b
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  Cigarette smoking impairs endothelial function. Hydroxymethylglutaryl (HMG) CoA reductase inhibitors (statins) may favorably affect endothelial function via nonlipid mechanisms. We tested the hypothesis that statins would improve endothelial function independent of changes in lipids in cigarette smokers. Twenty normocholesterolemic cigarette smokers and 20 matched healthy control subjects were randomized to atorvastatin 40 mg daily or placebo for 4 weeks, washed out for 4 weeks, and then crossed-over to the other treatment. Baseline low-density lipoprotein (LDL) levels were similar in smokers and healthy subjects, 103±22 versus 95±27 mg/dL, respectively (P=NS) and were reduced similarly in smokers and control subjects by atorvastatin, to 55±30 and 58±20 mg/dL, respectively (P=NS). Vascular ultrasonography was used to determine brachial artery, flow-mediated, endothelium-dependent, and nitroglycerin-mediated, endothelium-independent vasodilation. To elucidate potential molecular mechanisms that may account for changes in endothelial function, skin biopsy specimens were assayed for eNOS mRNA, eNOS activity, and nitrotyrosine. Endothelium-dependent vasodilation was less in smokers than nonsmoking control subjects during placebo treatment, 8.0±0.6% versus 12.1±1.1%, (P= 0.003). Atorvastatin increased endothelium-dependent vasodilation in smokers to 10.5±1.3% (P=0.017 versus placebo) but did not change endothelium-dependent vasodilation in control subjects (to 11.0±0.8%, P=NS). Endothelium-independent vasodilation did not differ between groups during placebo treatment and was not significantly affected by atorvastatin. Multivariate analysis did not demonstrate any association between baseline lipid levels or the change in lipid levels and endothelium-dependent vasodilation. Cutaneous nitrotyrosine levels and skin microvessel eNOS mRNA, but not ENOS activity, were increased in smokers compared with controls but unaffected by atorvastatin treatment. Atorvastatin restores endothelium-dependent vasodilation in normocholesterolemic cigarette smokers independent of changes in lipids. These results are consistent with a lipid-independent vascular benefit of statins but could not be explained by changes in eNOS message and tissue oxidative stress. These findings implicate a potential role for statin therapy to restore endothelial function and thereby investigate vascular disease in cigarette smokers.
• Liao, J. (2004). The JIM interview. Journal of Investigative Medicine, 52(Issue 6).
• Liao, J. K. (2004). Non-genomic activation of protein kinase Akt by steroid hormones. International Congress Series. doi:10.1016/j.ics.2003.12.059
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  Many cellular responses to steroid hormones involve the transcriptional modulation of target genes by the prototypical nuclear hormone receptor. In the classic model of steroid hormone action, the glucocorticoid receptor (GR) acts as ligand-dependent transcription factor by either activating or repressing gene expression through direct interactions with DNA or other transcription factors. Recent evidence suggests an important role for non-transcriptional effects of GR in the vascular system. The non-transcriptional actions of GR involve the rapid activation of protein kinases, such as phosphatidylinositol-3 kinase and Akt, leading to the activation of endothelial nitric oxide synthase (eNOS). This novel pathway of steroid hormone action protects against ischemic injury augmenting blood flow and decreasing vascular inflammation. © 2004, Elsevier Inc. All rights reserved.
• Liao, J. K. (2004). Statin therapy: Having the good without the bad. Hypertension, 43(Issue 6). doi:10.1161/01.hyp.0000126153.80112.5c
• Mital, S., & Liao, J. K. (2004). Statins and the myocardium. Seminars in Vascular Medicine, 4(Issue 4). doi:10.1055/s-2004-869594
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  Cardiac hypertrophy and heart failure are leading causes of morbidity and mortality worldwide. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, have been shown to inhibit cardiac hypertrophy and improve symptoms of heart failure by cholesterol-independent mechanisms. Statins block the isoprenylation and function of members of the Rho GTPase family, such as Rac1 and RhoA. Because Rac1 is a requisite component of NADPH oxidase, which is a major source of reactive oxygen species in cardiovascular cells, the ability of statins to inhibit Rac1-mediated oxidative stress contributes importantly to their inhibitory effects on cardiac hypertrophy. Furthermore, inhibition of RhoA by statins leads to the activation of protein kinase B/Akt and upregulation of Type 3 nitric oxide synthase in the endothelium and the heart. This activation and upregulation results in increased angiogenesis and myocardial perfusion, decreased myocardial apoptosis, and improvement in endothelial and cardiac function. Because these effects of statins occur independent of cholesterol lowering, statins may have therapeutic benefits in nonhyperlipidemic patients with cardiac hypertrophy and heart failure. Copyright © 2004 by Thieme Medical Publishers, Inc.
• Spiecker, M., Darius, H., Hankeln, T., Soufi, M., Sattler, A. M., Schaefer, J. R., Node, K., Börgel, J., Mügge, A., Lindpaintner, K., Huesing, A., Maisch, B., Zeldin, D. C., & Liao, J. K. (2004). Risk of coronary artery disease associated with polymorphism of the cytochrome P450 epoxygenase CYP2J2. Circulation, 110(Issue 15). doi:10.1161/01.cir.0000143832.91812.60
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  Background - Cytochrome P450 (CYP) 2J2 is expressed in the vascular endothelium and metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs). The EETs are potent endogenous vasodilators and inhibitors of vascular inflammation. However, it is not known whether genetic polymorphisms of CYP2J2 are associated with increased cardiovascular risks. Methods and Results - All 9 exons of the CYP2J2 gene and its proximal promoter were sequenced in 132 patients to identify potential variants. Functional consequence of a single nucleotide polymorphism (SNP) in the promoter of CYP2J2 was further evaluated by use of transcription factor-binding and reporter assays. A total of 17 polymorphisms were identified. One of the most relevant polymorphisms in terms of frequency and functional importance is located at -50 (G-50T) in the proximal promoter of CYP2J2. Screening of 289 patients with coronary artery disease and 255 control subjects revealed 77 individuals with the G-50T SNP (17.3% of coronary artery disease patients, 10.6% of control subjects; P=0.026). The association of the G-50T polymorphism remained significant after adjustment for age, gender, and conventional cardiovascular risk factors (OR, 2.23; 95% CI, 1.04 to 4.79). The G-50T mutation resulted in the loss of binding of the Sp1 transcription factor to the CYP2J2 promoter and resulted in a 48.1±2.4% decrease in CYP2J2 promoter activity (P
• Sun, J., & Liao, J. K. (2004). Induction of angiogenesis by heat shock protein 90 mediated by protein kinase Akt and endothelial nitric oxide synthase. Arteriosclerosis, Thrombosis, and Vascular Biology, 24(Issue 12). doi:10.1161/01.atv.0000147894.22300.4c
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  Objective - A specific inhibitor of heat shock protein 90 (Hsp90), 17-AAG, has been shown to inhibit tumor growth through cell cycle arrest, differentiation, or apoptosis. Because angiogenesis is important for tumor growth, we hypothesize that inhibition of angiogenesis by 17-AAG may mediate some of its antitumor effects. Methods and Results - Because protein kinase Akt and endothelial nitric oxide synthase (eNOS) are critical for angiogenesis, we studied the effects of 17-AAG on the phosphorylation and expression of Akt and eNOS in human umbilical vein endothelial cells. In a concentration- and time-dependent manner, inhibition of Hsp90 by 17-AAG decreased Akt and eNOS expression by 74% and 81%, respectively. Inhibition of eNOS expression by 17-AAG occurred at the transcriptional level as determined by eNOS promoter activity and nuclear run-on assay. Furthermore, treatment with 17-AAG decreased basal and vascular endothelial growth factor-stimulated Akt and eNOS phosphorylation. This corresponded with decreased NO production and inhibition of endothelial cell migration and angiogenesis. The anti-angiogenic effect of 17-AAG was partially reversed by the NO donor, SNAP. Conclusions - These findings indicate that Hsp90 is important not only for Akt and eNOS phosphorylation but also for eNOS gene transcription and suggests that Hsp90 may be a novel target for anti-angiogenic therapy.
• Wolfrum, S., Dendorfer, A., Rikitake, Y., Stalker, T. J., Gong, Y., Scalia, R., Dominiak, P., & Liao, J. K. (2004). Inhibition of Rho-kinase leads to rapid activation of phosphatidylinositol 3-kinase/protein kinase Akt and cardiovascular protection. Arteriosclerosis, Thrombosis, and Vascular Biology, 24(Issue 10). doi:10.1161/01.atv.0000142813.33538.82
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  Objective - Rho-Kinase activity is increased in cardiovascular diseases and in patients with cardiovascular risk factors. However, it is not known whether inhibition of Rho-kinase could lead to cardiovascular protection and, if so, by what mechanism. Methods and Results - In human endothelial cells, the Rho-kinase inhibitor, hydroxyfasudil (HF) (1 to 100 μmol/L), increased Akt serine-473 phosphorylation within 15 minutes, leading to a 2.2-fold and 4.0-fold increase in Akt kinase activity and nitric oxide (NO) release, respectively. Activation of Akt and eNOS by HF was completely blocked by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002 (10 μmol/L). To determine the physiological relevance of this pathway, we used 2 models of ischemia-reperfusion (I/R) injury. Acute administration of fasudil (10 mg/kg, intraperitoneal, 1 hour before ischemia) decreased leukocyte recruitment and adhesion to the mesenteric endothelium after I/R injury in wild-type but not eNOS-/- mice. Similarly, treatment with fasudil decreased myocardial infarct size by 38% in rats subjected to transient coronary artery occlusion. Cotreatment with 2 PI3-kinase inhibitors, wortmannin and LY294002, or the eNOS inhibitor, L-NAME, blocked the cardiovascular protective effects of fasudil. Conclusions - Inhibition of Rho-kinase leads to the activation of the PI3-kinase/Akt/eNOS pathway and cardiovascular protection. These findings suggest that Rho-kinase may play an important role in mediating the inflammatory response to I/R injury.
• Falck, J. R., Reddy, L. M., Reddy, Y. K., Bondlela, M., Krishna, U. M., Ji, Y., Sun, J., & Liao, J. K. (2003). 11,12-Epoxyeicosatrienoic acid (11,12-EET): Structural determinants for inhibition of TNF-α-induced VCAM-1 expression. Bioorganic and Medicinal Chemistry Letters, 13(Issue 22). doi:10.1016/j.bmcl.2003.08.060
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  A series of 11,12-EET analogues were synthesized and compared using a human endothelial cell based TNF-α-induced VCAM-1 expression assay. The resulting data were used to map a putative recognition/binding domain for 11,12-EET. © 2003 Elsevier Ltd. All rights reserved.
• Kawasaki, K., Smith, R. S., Hsieh, C. M., Sun, J., Chao, J., & Liao, J. K. (2003). Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis. Molecular and Cellular Biology, 23(Issue 16). doi:10.1128/mcb.23.16.5726-5737.2003
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  To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-L-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.
• Laufs, U., & Liao, J. K. (2003). Rapid effects of statins: From prophylaxis to therapy for ischemic stroke. Arteriosclerosis, Thrombosis, and Vascular Biology, 23(Issue 2). doi:10.1161/01.atv.0000051360.79309.4e
• Limbourg, F. P., Huang, Z., Plumier, J. C., Simoncini, T., Fujioka, M., Tuckermann, J., Schütz, G., Moskowitz, M. A., & Liao, J. K. (2003). Erratum: Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids (Journal of Clinical Investigation (2002) 110 (1729-1738)). Journal of Clinical Investigation, 111(Issue 5). doi:10.1172/jci200315737e1
• Nakagami, H., Takemoto, M., & Liao, J. K. (2003). NADPH oxidase-derived superoxide anion mediates angiotensin II-induced cardiac hypertrophy. Journal of Molecular and Cellular Cardiology, 35(Issue 7). doi:10.1016/s0022-2828(03)00145-7
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  Cardiac hypertrophy is an adaptive response to increases in blood pressure. Recent studies indicate that the hypertrophic process is associated with increases in intracellular oxidative stress in cardiomyocytes. We hypothesize that superoxide anion mediates the hypertrophic response and that antioxidant therapy may be effective in attenuating cardiac hypertrophy. Neonatal rat cardiac myocytes were stimulated with angiotensin II (AngII, 1 μM) with and without various antioxidants. N-acetylcysteine (NAC, 10 mM) and probucol (50 μM), and to a lesser extent, vitamin C (500 μM) and reduced glutathione (1 mM), inhibited AngII-induced [3H]-leucine uptake and atrial natriuretic factor (ANF) promoter activity. The hypertrophic response is mediated by superoxide anion (O2-·) since cell-permeable polyethylene glycol (PEG)-conjugated superoxide dismutase (50 U/ml), but not PEG-catalase (500 U/ml), attenuated AngII-induced [3H]-leucine uptake and ANF promoter activity. Furthermore, NAC blocked AngII-induced increase in myocardial oxidative stress, decreased the expression of ANF and myosin light chain-2v, and inhibited the re-organization of cytoskeletal proteins, desmin and α-actinin. These effects of AngII were abolished by angiotensin type 1 receptor blocker, losartan, but not type 2 receptor blocker, PD123319. Indeed, co-administration of losartan (10 mg/kg/d, 14 d) or NAC (200 mg/kg/d, 14 d) inhibited AngII-induced O2-· production and cardiac hypertrophy in rats without affecting blood pressure. These findings indicate that the generation of O2-· contributes to oxidant-induced hypertrophic response and suggest that antioxidant therapy may have beneficial effects in cardiac hypertrophy. © 2003 Elsevier Science Ltd. All rights reserved.
• Node, K., Fujita, M., Kitakaze, M., Hori, M., & Liao, J. K. (2003). Short-term statin therapy improves cardiac function and symptoms in patients with idiopathic dilated cardiomyopathy. Circulation, 108(Issue 7). doi:10.1161/01.cir.0000084539.58092.de
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  Background - Chronic heart failure is associated with inflammation and neurohormonal imbalance. The 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, or statins, exert anti-inflammatory and vascular protective effects. We hypothesized that short-term statin therapy may have beneficial effects in patients with nonischemic heart failure. Methods and Results - Sixty-three patients with symptomatic, nonischemic, dilated cardiomyopathy were randomly divided into 2 groups. One group received simvastatin (n = 24), and the other group received placebo (n = 27). The initial dose of simvastatin was 5 mg/d, which was increased to 10 mg/d after 4 weeks. After 14 weeks, patients receiving simvastatin exhibited a modest reduction in serum cholesterol level compared with patients receiving placebo (130±13 versus 148±18, P
• Prorock, A. J., Hafezi-Moghadam, A., Laubach, V. E., Liao, J. K., & Ley, K. (2003). Vascular protection by estrogen in ischemia-reperfusion injury requires endothelial nitric oxide synthase. American Journal of Physiology - Heart and Circulatory Physiology, 284(Issue 1). doi:10.1152/ajpheart.00957.2001
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  Estrogen increases nitric oxide (NO) production by inducing the activity of endothelial NO synthase (eNOS) (Simoncini et al. Nature 407: 538, 2000). Ischemia (30 min) and reperfusion (I/R) increased the number of adherent leukocytes and decreased their rolling velocities in mouse cremaster muscle venules with a strong dependence on wall shear rate. Minimum rolling velocity at ∼5 min after the onset of reperfusion was accompanied by increased P-selectin expression. This preceded the peak in leukocyte adhesion (at 10-15 min). In untreated wild-type mice, I/R caused a decrease of leukocyte rolling velocity from 37 to 26 μm/s and a 2.0-fold increase in leukocyte adhesion. Both were completely abolished by 0.25 mg ip estrogen I h before surgery. In eNOS-/- mice, the decrease of leukocyte rolling velocity and increase in adhesion were similar but were only marginally improved by estrogen. We conclude that the protective effect of estrogen, as measured by leukocyte rolling and adhesion, is significantly reduced in eNOS-/- mice, suggesting that induction of eNOS activity is the major mechanism of vasoprotection by estrogen in this model.
• Simoncini, T., Rabkin, E., & Liao, J. K. (2003). Molecular basis of cell membrane estrogen receptor interaction with phosphatidylinositol 3-kinase in endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology, 23(Issue 2). doi:10.1161/01.atv.0000053846.71621.93
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  Objective - Nontranscriptional signaling mechanisms mediate some of the biological effects of estrogen, such as the rapid actions on the blood vessels. By interacting with phosphatidylinositol 3-kinase (PI3K), estrogen receptor (ER) α leads to activation of protein kinase Akt and to subsequent increase in endothelial nitric oxide synthase activity. Because PI3K is mainly a cytoplasmic complex, we studied the cellular site of interaction between this enzyme and ERα, and we dissected the molecular mechanisms that mediate this interaction. Methods and Results - By using cultured human saphenous vain endothelial cells, we found that cell membrane-bound ERα colocalizes with PI3K and may be responsible for PI3K activation. Furthermore, we characterized the subsequent steps in the activation of the PI3K/Akt signaling cascade, comparing the molecular events that follow insulin or estradiol activation of PI3K. Conclusions - We provide novel evidence for an important role of nonnuclear estrogen receptor in rapid, nontranscriptional responses of human endothelial cells to estrogen.
• Trochu, J. N., Mital, S., Zhang, X. P., Xu, X., Ochoa, M., Liao, J. K., Recchia, F. A., & Hintze, T. H. (2003). Preservation of NO production by statins in the treatment of heart failure. Cardiovascular Research, 60(Issue 2). doi:10.1016/j.cardiores.2003.08.003
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  Objective: Because statins promote endogenous nitric oxide (NO) production in vessels by increasing endothelial nitric oxide synthase (eNOS), we evaluated the clinical benefit and efficiency of simvastatin in preventing the decrease in NO control of coronary blood flow (CBF), NO regulation of myocardial oxygen consumption (MVO2) and decreased nitrite production in coronary microvessels, associated with pacing-induced heart failure (HF). Methods: Dogs (n=17) were instrumented for measurement of coronary blood flow and left ventricular end diastolic pressure (LVEDP). HF was induced by pacing. Ten dogs were given simvastatin 20 mg/kg/day orally (HF+SIMVA) from the 10th day of pacing. Results: HF+SIMVA had a lower LVEDP at 4 weeks of pacing (18±1 vs. 25±1 mm Hg, p
• Wolfrum, S., Grimm, M., Heidbreder, M., Dendorfer, A., Katus, H. A., Liao, J. K., & Richardt, G. (2003). Acute reduction of myocardial infarct size by a hydroxymethyl glutaryl coenzyme a reductase inhibitor is mediated by endothelial nitric oxide synthase. Journal of Cardiovascular Pharmacology, 41(Issue 3). doi:10.1097/00005344-200303000-00017
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  In addition to their lipid-lowering properties, statins improve endothelial function by increasing the activity of endothelial nitric oxide synthase (eNOS). It was hypothesized that, by this mechanism, statins protect the myocardium from ischemia/reperfusion injury in normocholesterolemic animals. Rats were pretreated for 1 week with either cerivastatin (0.3 mg/kg/d) or placebo. Anesthetized animals underwent 30 minutes of coronary artery occlusion (CAO) followed by 180 minutes of reperfusion. In a separate set of experiments, the NOS inhibitor L-NAME (15 mg/kg; Nω-nitro-L-arginine methyl ester) was administered 15 minutes before CAO. Cerivastatin decreased infarct size by 49% (P < 0.05) without reducing plasma cholesterol levels. Cerivastatin increased myocardial eNOS mRNA and NOS activity and by 52% and 58% (P < 0.05), respectively. Cardioprotection and upregulation of eNOS activity evoked by cerivastatin were not observed in rats cotreated with L-NAME. These results show that statins reduce the extent of myocardial necrosis in normocholesterolemic rats after acute ischemia/reperfusion injury by increasing myocardial eNOS activity. Therefore, statins may protect the heart not only by reducing the incidence of ischemic events, but also by limiting cell damage during acute myocardial infarction.
• Grimm, M., Spiecker, M., De Caterina, R., Shin, W. S., & Liao, J. K. (2002). Inhibition of major histocompatibility complex class II gene transcription by nitric oxide and antioxidants. Journal of Biological Chemistry, 277(Issue 29). doi:10.1074/jbc.m110538200
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  Interferon (IFN)-γ facilitates cellular immune response, in part, by inducing the expression of major histocompatibility complex class II (MHC-II) molecules. We demonstrate that IFN-γ induces the expression of HLA-DRA in vascular endothelial cells via mechanisms involving reactive oxygen species. IFN-γ-induced HLA-DRA expression was inhibited by nitric oxide (NO) and antioxidants such as superoxide dismutase, catalase, pyrrolidine dithiocarbamate, and N-acetylcysteine. Nuclear run-on assays demonstrated that NO and antioxidants inhibited IFN-γ-induced HLA-DRA gene transcription. Transient transfection studies using a fully functional HLA-DRA promoter construct ([-300]-DRα.CAT) showed that inhibition of endogenous NO synthase activity by Nω-monomethyl-L-arginine or addition of exogenous hydrogen peroxide (H2O2) augmented basal and IFN-γ-stimulated [-300] DRα.CAT activity. However, H2O2 and Nω-monomethyl-L-arginine could induce HLA-DRA expression suggesting that H2O2 is a necessary but not a sufficient mediator of IFN-γ-induced HLA-DRA expression. Electrophoretic mobility shift assay and Western blotting demonstrated that NO and antioxidants had little or no effect on IFN-γ-induced IRF-1 activation or MHC-II transactivator (CIITA) expression but did inhibit IFN-γ-induced activation of STAT1α (p91) and Y box transcription factors, NF-YA and NF-YB. These results indicate that NO and antioxidants may attenuate vascular inflammation by antagonizing the effects of intracellular reactive oxygen species generation by IFN-γ, which is necessary for MHC-II gene transcription.
• Hafezi-Moghadam, A., Simoncini, T., Yang, Z., Limbourg, F. P., Plumier, J. C., Rebsamen, M. C., Hsieh, C. M., Chui, D. S., Thomas, K. L., Prorock, A. J., Laubach, V. E., Moskowitz, M. A., French, B. A., Ley, K., & Liao, J. K. (2002). Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase. Nature Medicine, 8(Issue 5). doi:10.1038/nm0502-473
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  Corticosteroids have been shown to exert beneficial effects in the treatment of acute myocardial infarction, but the precise mechanisms underlying their protective effects are unknown. Here we show that high-dose corticosteroids exert cardiovascular protection through a novel mechanism involving the rapid, non-transcriptional activation of endothelial nitric oxide synthase (eNOS). Binding of corticosteroids to the glucocorticoid receptor (GR) stimulated phosphatidylinositol 3-kinase and protein kinase Akt, leading to eNOS activation and nitric oxide-dependent vasorelaxation. Acute administration of pharmacological concentrations of corticosteroids in mice led to decreased vascular inflammation and reduced myocardial infarct size following ischemia and reperfusion injury. These beneficial effects of corticosteroids were abolished by GR antagonists or eNOS inhibitors in wild-type mice and were completely absent in eNOS-deficient (Nos3−/−) mice. The rapid activation of eNOS by the non-nuclear actions of GR, therefore, represents an important cardiovascular protective effect of acute high-dose corticosteroid therapy.
• Ho, K. J., & Liao, J. K. (2002). Nonnuclear actions of estrogen. Arteriosclerosis, Thrombosis, and Vascular Biology, 22(Issue 12). doi:10.1161/01.atv.0000041200.85946.4a
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  Estrogen has long been observed to endow cardiovascular protective effects, as evidenced by sex-specific differences in the incidence of hypertensive and coronary artery disease, the development of atherosclerosis, and myocardial remodeling after infarction. To exert its tissue-specific effects, the classic estrogen receptor (ER) functions as a ligand-dependent transcription factor. However, there is growing evidence that in response to 17β-estradiol and heterologous signals, the ER can also mediate signaling cascades at the membrane and in the cytoplasm via various second messengers, such as receptor-mediated protein kinases. This review summarizes the current understanding of nonnuclear ER signaling and discusses the relevance to eliciting the beneficial cardiovascular effects of estrogen. These include vasodilation, inhibition of response to vessel injury, limiting myocardial injury after infarction, and attenuating cardiac hypertrophy. Defining the full repertoire of ER function promises to expose novel, highly specific targets for pharmacological interventions and may ultimately lead to the primary and secondary prevention of cardiovascular diseases.
• Liao, J. K. (2002). Shedding growth factors in cardiac hypertrophy. Nature Medicine, 8(Issue 1). doi:10.1038/nm0102-20
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  The finding that cleavage and shedding of the membrane-bound heparin-binding epidermal growth factor by metalloproteases contribute to the hypertrophic process offers new insights for the treatment of cardiac hypertrophy and progression to heart failure. (pages 35-40).
• Limbourg, F. P., Huang, Z., Plumier, J. C., Simoncini, T., Fujioka, M., Tuckermann, J., Schütz, G., Moskowitz, M. A., & Liao, J. K. (2002). Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids. Journal of Clinical Investigation, 110(Issue 11). doi:10.1172/jci0215481
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  Many cellular responses to corticosteroids involve the transcriptional modulation of target genes by the glucocorticoid receptor (GR). A rapid, non-nuclear effect of GR was found to mediate neuroprotection. High-dose corticosteroids (20 mg/kg intraperitoneally), given within 2 hours of transient cerebral ischemia, acutely increased endothelial nitric oxide synthase (eNOS) activity, augmented regional cerebral blood flow (CBF) by 40% to 50%, and reduced cerebral infarct size by 32%. These neuroprotective effects of corticosteroids were abolished by the GR antagonist RU486 and by inhibition of phosphatidylinositol 3-kinase (PI3K), and were absent in eNOS-/- mice. To determine the mechanism by which GR activated eNOS, we measured the effect of corticosteroids on PI3K and the protein kinase Akt. In a ligand-dependent manner, GR activated PI3K and Akt in vitro and in vivo caused NO-dependent vasodilation, which was blocked by cotreatment with RU486 or the PI3K inhibitor LY294002 but not by transcriptional inhibitors. Indeed, a mutant GR, which cannot dimerize and bind to DNA, still activated PI3K and Akt in response to corticosteroids. These findings indicate that non-nuclear GR rapidly activates eNOS through the PI3K/Akt pathway and suggest that this mechanism mediates the acute neuroprotective effects of corticosteroids through augmentation of CBF.
• Rebsamen, M. C., Sun, J., Norman, A. W., & Liao, J. K. (2002). 1α,25-dihydroxyvitamin D3 induces vascular smooth muscle cell migration via activation of phosphatidylinositol 3-kinase. Circulation Research, 91(Issue 1). doi:10.1161/01.res.0000025269.60668.0f
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  The steroid hormone aα,25-dihydroxyvitamin D3 [1α,25-(OH)2D3] promotes vascular smooth muscle cell (VSMC) growth and calcification, but the precise mechanism by which 1α,25-(OH)2D3 regulates VSMC migration is unknown. In rat aortic SMCs, we found that 1α,25-(OH)2D3 (0.1 to 100 nmol/L) induced a dose-dependent increase in VSMC migration. This response required the activation of phosphatidylinositol 3-kinase (PI3 kinase) because 1α,25-(OH)2D3-induced migration was completely abolished by the PI3 kinase inhibitors, LY294002 (10 μmol/L) or wortmannin (30 nmol/L). Furthermore, the RNA polymerase inhibitor, 5,6-dichlorobenzimidazole riboside (50 μmol/L), did not affect 1α,25-(OH)2D3-induced VSMC migration, suggesting that gene transcription is not involved in this rapid response. Using analogs of 1α,25-(OH)2D3, which have been characterized for their abilities to induce either transcriptional or nontranscriptional responses of 1α,25-(OH)2D3, we found that 1α,25-dihydroxylumisterol, which is a potent agonist of the rapid, nongenomic responses, was equipotent with 1α,25-(OH)2D3 in inducing PI3 kinase activity and VSMC migration. Moreover, 1β,25-(OH)2D3, which specifically antagonizes the nongenomic actions of 1α,25-(OH)2D3, abolished 1α,25-(OH)2D3-induced PI3 kinase activity and VSMC migration, whereas the inhibitor of the genomic actions of vitamin D, (23S)-25-dehydro-1α-OH-D3-26,23-lactone, did not affect these responses. These results indicate that 1α,25-(OH)2D3 induces VSMC migration independent of gene transcription via PI3 kinase pathway, and suggest a possible mechanism by which 1α,25-(OH)2D3 may contribute to neointima formation in atherosclerosis and vascular remodeling.
• Sakata, Y., Kamei, C. N., Nakagami, H., Bronson, R., Liao, J. K., & Chin, M. T. (2002). Ventricular septal defect and cardiomyopathy in mice lacking the transcription factor CHF1/HEY2. Proceedings of the National Academy of Sciences of the United States of America, 99(Issue 25). doi:10.1073/pnas.252648999
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  Ventricular septal defects are common in human infants, but the genetic programs that control ventricular septation are poorly understood. Here we report that mice with a targeted disruption of the cardiovascular basic helix-loop-helix factor (CHF)1/Hey2 gene show isolated ventricular septal defects. These defects result primarily in failure to thrive. Mice often succumbed within the first 3 wk after birth and showed pulmonary and liver congestion. The penetrance of this phenotype varied, depending on genetic background, suggesting the presence of modifier genes. Expression patterns of other cardiac-specific genes were not affected. Of the few animals on a mixed genetic background that survived to adulthood, most developed a cardiomyopathy but did not have ventricular septal defects. Our results indicate that CHF1 plays an important role in regulation of ventricular septation in mammalian heart development and is important for normal myocardial contractility. These mice provide a useful model for the study of the ontogeny and natural history of ventricular septal defects and cardiomyopathy.
• Simoncini, T., Fornari, L., Mannella, P., Varone, G., Caruso, A., Liao, J. K., & Genazzani, A. R. (2002). Novel non-transcriptional mechanisms for estrogen receptor signaling in the cardiovascular systemInteraction of estrogen receptor α with phosphatidylinositol 3-OH kinase. Steroids, 67(Issue 12). doi:10.1016/s0039-128x(02)00040-5
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  Estrogen receptor (ER) signaling has been, for a long time, associated with transcriptional processes involving nuclear translocation and binding on specific response elements, leading to regulation of target gene expression. However, rapid, non-transcriptional mechanisms of signal transduction through steroid hormone receptors have been identified. These so-called 'non-genomic' effects are independent from gene transcription or protein synthesis and involve steroid-induced modulation of cytoplasmic or cell membrane-bound regulatory proteins. Several biological actions of estrogen have been associated with this type of signaling, and intracellular regulatory cascades such as extracellular signal-regulated kinase/mitogen-activated protein kinases (ERK/MAPK) and tyrosine kinases or the modulation of G-protein-coupled receptors have been shown to be non-transcriptionally recruited by estrogen in diverse tissues. The vascular wall is one of these sites, where estrogen triggers rapid vasodilatation mainly due to increased nitric oxide (NO) release. We have recently described a novel, non-transcriptional mechanism for ER signaling in human as well as in animal endothelial cells, showing that ERα can physically and functionally couple to the lipid kinase phosphatidylinositol 3-OH kinase (PI3K). This interaction leads to activation of PI3K signaling cascade to Ser/Thr kinase Akt, which mediates several PI3K-dependent intracellular effects, including endothelial isoform of NO synthase (eNOS) phosphorylation and activation. This original non-transcriptional mechanism for ER signaling may play an important role in the generation of some of the rapid 'non-genomic' effects of estrogen. Copyright © 2002 Elsevier Science Inc.
• Simoncini, T., Genazzani, A. R., & Liao, J. K. (2002). Nongenomic mechanisms of endothelial nitric oxide synthase activation by the selective estrogen receptor modulator raloxifene. Circulation, 105(Issue 11). doi:10.1161/hc1102.105267
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  Background - Nontranscriptional signaling through estrogen receptors (ERs) is important in the cardiovascular system. In particular, estrogen stimulates endothelial NO synthase (eNOS) via the phosphatidylinositol 3-kinase (PI3K) pathway. The selective estrogen receptor modulator (SERM) raloxifene is effective for the treatment of postmenopausal osteoporosis, but its ability to activate eNOS via PI3K is unknown. Methods and Results - Human umbilical vein endothelial cells were cultured in estrogen-deprived, phenol red-free medium. Raloxifene stimulated eNOS in a concentration- and time-dependent manner. Activation of eNOS by raloxifene was blocked by the PI3K inhibitor wortmannin and by the ER antagonist ICI 182,780 but not by transcriptional or translational inhibitors. Coimmunoprecipitation studies demonstrated that, in a ligand-dependent manner, raloxifene increased ERα-associated p85α, p 110α, and PI3K activity. This correlated temporally with increases in the serine and threonine phosphorylation and activation of protein kinase Akt. Conclusions - Our findings indicate that nongenomic ER signaling triggered by a SERM leads to a rapid activation of NO synthesis in human endothelial cells. The ability of raloxifene to facilitate ERα-PI3K interaction may provide additional insight into the structure-function relationship of specific SERMs, which promote the nontranscriptional effects of ER.
• Sun, J., & Liao, J. K. (2002). Functional interaction of endothelial nitric oxide synthase with a voltage-dependent anion channel. Proceedings of the National Academy of Sciences of the United States of America, 99(Issue 20). doi:10.1073/pnas.202260999
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  Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS) whose function is modulated, in part, by specific protein interactions. By coimmunoprecipitation experiments followed by MS analyses, we identified a human voltage-dependent anion/cation channel or porin as a binding partner of eNOS. The interaction between porin and eNOS was demonstrated by coimmunoprecipitation studies in nontransfected human endothelial cells and Cos-7 cells transiently transfected with eNOS and porin cDNAs. In vitro binding studies with glutathione S-transferase-porin indicated that porin binds directly to eNOS and that this interaction augmented eNOS activity. The calcium ionophore, A23187, and bradykinin, which are known to activate eNOS, markedly increased porin-eNOS interaction, suggesting a potential role of intracellular Ca2+ in mediating this interaction. Theses results indicate that the interaction between a voltage-dependent membrane channel and eNOS may be important for regulating eNOS activity.
• Sun, J., Sui, X. X., Bradbury, J. A., Zeldin, D. C., Conte, M. S., & Liao, J. K. (2002). Inhibition of vascular smooth muscle cell migration by cytochrome P450 epoxygenase-derived eicosanoids. Circulation Research, 90(Issue 9). doi:10.1161/01.res.0000017727.35930.33
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  Vascular smooth muscle cell (SMC) migration and proliferation contribute to neointimal hyperplasia and restenosis after vascular injury. The epoxyeicosatrienoic acids (EETs), which are products of cytochrome P450 (CYP) epoxygenases, possess vasodilatory, antiinflammatory, and fibrinolytic properties. To determine whether these compounds also possess antimigratory and antiproliferative properties, we stimulated rat aortic SMCs with either 20% serum or platelet-derived growth factor (PDGF-BB, 20 ng/mL). In a concentration-dependent manner, treatment with EETs, particularly 11,12-EET, inhibited SMC migration through a modified transwell filter by 53% to 60%. EETs, however, have no inhibitory effects on PDGF-stimulated SMC proliferation. Adenoviral-mediated overexpression of the CYP isoform, CYP2J2, in SMCs also inhibited serum- and PDGF-induced SMC migration by 32% and 26%, respectively; both effects of which were reversed by the CYP inhibitors SKF525A or clotrimazole, but not by the KCa channel blocker, charybdotoxin, or the cyclooxygenase inhibitor, diclofenac. The effect of EETs correlated with increases in intracellular cAMP levels. Indeed, forskolin and 8-bromo-cAMP exert similar inhibitory effects on SMC migration as 11,12-EET and the effects of 11,12-EET were blocked by cAMP and protein kinase A (PKA) inhibitors. These findings indicate that EETs possess antimigratory effects on SMCs through the cAMP-PKA pathway and suggest that CYP epoxygenase-derived eicosanoids may play important roles in vascular disease and remodeling.
• Takemoto, M., Sun, J., Hiroki, J., Shimokawa, H., & Liao, J. K. (2002). Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation, 106(Issue 1). doi:10.1161/01.cir.0000020682.73694.ab
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  Background - Hypoxia-induced pulmonary hypertension is a major cause of morbidity and mortality. Hypoxia induces pulmonary vasoconstriction, in part, by decreasing endothelial nitric oxide synthase (eNOS) expression. The mechanism by which hypoxia decreases eNOS expression is not known but may involve Rho-kinase-induced actin cytoskeletal changes in vascular endothelial cells. Methods and Results - To determine whether hypoxia regulates eNOS expression through Rho-kinase, we exposed human saphenous and pulmonary artery endothelial cells to hypoxia (3% O2) with and without a Rho-kinase inhibitor, hydroxyfasudil (0.1 to 100 μmol/L), for various durations (0 to 48 hours). Hypoxia increased Rho-kinase expression and activity by 50% and 74%, decreased eNOS mRNA and protein expression by 66±3% and 57±5%, and inhibited eNOS activity by 48±9%. All of these effects of hypoxia on eNOS were reversed by cotreatment with hydroxyfasudil. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase or Rho-kinase by overexpression of dominant-negative Rho-kinase reversed hypoxia-induced decrease in eNOS expression. Indeed, disruption of the actin cytoskeleton, the downstream target of Rho-kinase, by cytochalasin D also upregulated eNOS expression. Hypoxia reduced eNOS mRNA half-life from 22±2 to 13±2 hours, which was reversed by cotreatment with hydroxyfasudil. However, neither hypoxia nor hydroxyfasudil had any effects on eNOS gene transcription. Conclusions - These results indicate that hypoxia-induced decrease in eNOS expression is mediated by Rho-kinase and suggest that Rho-kinase inhibitors may have therapeutic benefits in patients with hypoxia-induced pulmonary hypertension.
• Amin-Hanjani, S., Stagliano, N. E., Yamada, M., Huang, P. L., Liao, J. K., & Moskowitz, M. A. (2001). Mevastatin, an HMG-CoA reductase inhibitor, reduces stroke damage and upregulates endothelial nitric oxide synthase in mice. Stroke, 32(Issue 4). doi:10.1161/01.str.32.4.980
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  Background and Purpose - The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) lower serum cholesterol and decrease the incidence of stroke and cardiovascular disease. There is growing evidence that statins exert some of their beneficial effects independent of cholesterol lowering. Indeed, we have previously demonstrated that chronic simvastatin administration upregulates endothelial nitric oxide synthase (eNOS), resulting in more functional protein, augmentation of cerebral blood flow, and neuroprotection in a murine model of cerebral ischemia. In this report we examined whether another member of the statin family shared these effects and whether eNOS upregulation is sustained with longer treatment. Methods - Mevastatin (2 mg/kg or 20 mg/kg per day) was administered to 18- to 22-g male mice for 7, 14, or 28 days before 2-hour middle cerebral artery occlusion with the use of the filament model (n=9 to 12). Neurological deficits and cerebral infarct volumes were assessed at 24 hours. Arterial blood pressure and gases, relative cerebral blood flow, and blood cholesterol levels were monitored in a subset of animals (n=5). Absolute cerebral blood flow was measured by the [14C]iodoamphetamine indicator fractionation technique (n=6). eNOS mRNA and protein levels were determined. Results - Mevastatin increased levels of eNOS mRNA and protein, reduced infarct size, and improved neurological deficits in a dose- and time-dependent manner. Greatest protection was seen with 14- and 28-day high-dose treatment (26% and 37% infarct reduction, respectively). Cholesterol levels were reduced only after 28 days of treatment and did not correlate with infarct reduction. Baseline absolute cerebral blood flow was 30% higher after 14-day high-dose treatment. Conclusions - Chronic prophylactic treatment with mevastatin upregulated eNOS and augmented cerebral blood flow. These changes occurred in the absence of changes in serum cholesterol levels, were sustained for up to 1 month of treatment, and resulted in neuroprotection after middle cerebral artery occlusion.
• De Caterina, R., Bourcier, T., Laufs, U., La Fata, V., Lazzerini, G., Neish, A. S., Libby, P., & Liao, J. K. (2001). Induction of endothelial-leukocyte interaction by interferon-γ requires coactivation of nuclear factor-κB. Arteriosclerosis, Thrombosis, and Vascular Biology, 21(Issue 2). doi:10.1161/01.atv.21.2.227
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  To determine whether nuclear factor (NF)-κB is necessary to confer endothelial cell responsiveness to interferon (INF)-γ in terms of vascular cell adhesion molecule (VCAM)-1 expression and leukocyte adhesion, human endothelial cells were treated with IFN-γ in the presence of low concentrations (LCs) of interleukin (IL)-1α (≤ 100 pg/mL), which activates NF-κB but does not induce VCAM-1 expression. Although IFN-γ induced major histocompatibility complex class II antigen expression and although a high concentration of IL-1α (10 ng/mL) induced leukocyte adhesion and VCAM-1 expression, neither IFN-γ nor LC IL-1α was able to induce VCAM-1 expression or leukocyte adhesion. However, the combination of IFN-γ and LC IL-1α induced VCAM-1 expression and increased leukocyte adhesion (67% and 49% of high-concentration IL-1α, respectively). Electrophoretic mobility shift assays and immunoblotting of nuclear extracts showed that IFN-γ activated signal transducers and activators of transcription (STAT)-1α and interferon regulatory factor (IRF)-1 but not NF-κB, whereas LC IL-1α activated NF-κB but not STAT-1α or IRF-1. Nuclear run-on studies showed that LC IL-1α is necessary but not sufficient for inducing VCAM-1 gene transcription and that the combination of IFN-γ and LC IL-1α is required for full VCAM-1 gene transcription. These findings suggest that factors that activate NF-κB can synergize with IFN-γ in promoting endothelial-leukocyte interaction.
• Node, K., Ruan, X. L., Dai, J., Yang, S. X., Graham, L. R., Zeldin, D. C., & Liao, J. K. (2001). Activation of Gαs Mediates Induction of Tissue-type Plasminogen Activator Gene Transcription by Epoxyeicosatrienoic Acids. Journal of Biological Chemistry, 276(Issue 19). doi:10.1074/jbc.m100439200
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  The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 (CYP) epoxygenases that have vasodilatory and anti-inflammatory properties. Here we report that EETs have additional fibrinolytic properties. In vascular endothelial cells, physiological concentrations of EETs, particularly 11,12-EET, or overexpression of the endothelial epoxygenase, CYP2J2, increased tissue plasminogen activator (t-PA) expression by 2.5-fold without affecting plasminogen activator inhibitor-1 expression. This increase in t-PA expression correlated with a 4-fold induction in t-PA gene transcription and a 3-fold increase in t-PA fibrinolytic activity and was blocked by the CYP inhibitor, SKF525A, but not by the calcium-activated potassium channel blocker, charybdotoxin, indicating a mechanism that does not involve endothelial cell hyperpolarization. The t-PA promoter is cAMP-responsive, and induction of t-PA gene transcription by EETs correlated with increases in intracellular cAMP levels and, functionally, with cAMP-driven promoter activity. To determine whether increases in intracellular cAMP levels were due to modulation of guanine nucleotide-binding proteins, we assessed the effects of EETs on Gαs and Gαi2. Treatment with EETs increased Gαs, but not Gαi2, GTP-binding activity by 3.5-fold. These findings indicate that EETs possess fibrinolytic properties through the induction of t-PA and suggest that endothelial CYP2J2 may play an important role in regulating vascular hemostasis.
• Sun, J., Kamei, C. N., Layne, M. D., Jain, M. K., Liao, J. K., Lee, M. E., & Chin, M. T. (2001). Regulation of Myogenic Terminal Differentiation by the Hairy-related Transcription Factor CHF2. Journal of Biological Chemistry, 276(Issue 21). doi:10.1074/jbc.m101163200
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  We have recently cloned a novel basic helix-loop-helix factor, CHF2, that functions as a transcriptional repressor. To address its role in the regulation of myogenic terminal differentiation, we analyzed its expression pattern during C2C12 mouse myotube formation. In undifferentiated myoblasts, CHF2 is expressed at high levels. After induction of myotube formation in low serum, CHF2 expression is barely detectable at 3 days after induction. Myogenin expression, in contrast, peaks at 3 days. In transiently transfected 10T1/2 embryonic fibroblasts, CHF2 inhibited MyoD-dependent activation of the myogenin promoter in a dose-dependent fashion. Electrophoretic mobility shift analysis indicated that CHF2 inhibits the binding of the MyoD·E47 heterodimer to the E-box binding site. CHF2 also inhibited myogenic conversion of 10T1/2 cells by MyoD, as measured by skeletal myosin heavy chain protein expression. Coimmunoprecipitation analysis indicated that CHF2 forms a protein complex with MyoD. Mutational analysis of CHF2 indicated that the repression activity for both transcription and myogenic conversion mapped to a hydrophobic carboxyl-terminal region and did not require either the basic helix-loop-helix or YRPW motifs. Our data indicate that CHF2 functions as a transcriptional repressor of myogenesis by formation of an inactive heterodimeric complex with MyoD and likely plays an important role in muscle development.
• Takemoto, M., & Liao, J. K. (2001). Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Arteriosclerosis, Thrombosis, and Vascular Biology, 21(Issue 11). doi:10.1161/hq1101.098486
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  The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or statins are potent inhibitors of cholesterol biosynthesis. Several large clinical trials have demonstrated the beneficial effects of statins in the primary and secondary prevention of coronary heart disease. However, the overall clinical benefits observed with statin therapy appear to be greater than what might be expected from changes in lipid profile alone, suggesting that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels. Indeed, recent experimental and clinical evidence indicates that some of the cholesterol-independent or "pleiotropic" effects of statins involve improving or restoring endothelial function, enhancing the stability of atherosclerotic plaques, and decreasing oxidative stress and vascular inflammation. Many of these pleiotropic effects of statins are mediated by their ability to block the synthesis of important isoprenoid intermediates, which serve as lipid attachments for a variety of intracellular signaling molecules. In particular, the inhibition of small GTP-binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation, may play an important role in mediating the direct cellular effects of statins on the vascular wall.
• Takemoto, M., Node, K., Nakagami, H., Liao, Y., Grimm, M., Takemoto, Y., Kitakaze, M., & Liao, J. K. (2001). Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. Journal of Clinical Investigation, 108(Issue 10). doi:10.1172/jci13350
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  Cardiac hypertrophy is a major cause of morbidity and mortality worldwide. The hypertrophic process is mediated, in part, by small G proteins of the Rho family. We hypothesized that statins, inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, inhibit cardiac hypertrophy by blocking Rho isoprenylation. We treated neonatal rat cardiac myocytes with angiotensin II (AngII) with and without simvastatin (Sim) and found that Sim decreased AngII-induced protein content, [3H] leucine uptake, and atrial natriuretic factor (ANF) promoter activity. These effects were associated with decreases in cell size, membrane Rho activity, superoxide anion [O2·-) production, and intracellular oxidation, and were reversed with L-mevalonate or geranylgeranylpyrophosphate, but not with farnesylpyrophosphate or cholesterol. Treatments with the Rho inhibitor C3 exotoxin and with cell-permeable superoxide dismutase also decreased AnglI-induced O2·- production and myocyte hypertrophy. Overexpression of the dominant-negative Rho mutant N17Rac1 completely inhibited AngII-induced intracellular oxidation and ANF promoter activity, while N19RhoA partially inhibited it, and N17Cdc42 had no effect. Indeed, Sim inhibited cardiac hypertrophy and decreased myocardial Rac1 activity and O2·- production in rats treated with AngII infusion or subjected to transaortic constriction. These findings suggest that statins prevent the development of cardiac hypertrophy through an antioxidant mechanism involving inhibition of Rac1.
• Yang, B., Graham, L., Dikalov, S., Mason, R. P., Falck, J. R., Liao, J. K., & Zeldin, D. C. (2001). Overexpression of cytochrome P450 CYP2J2 protects against hypoxia-reoxygenation injury in cultured bovine aortic endothelial cells. Molecular Pharmacology, 60(Issue 2). doi:10.1124/mol.60.2.310
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  CYP2J2 is abundant in human heart and its arachidonic acid metabolites, the epoxyeicosatrienoic acids (EETs), have potent vasodilatory, antiinflammatory and cardioprotective properties. This study was designed to examine the role of CYP2J2 in hypoxia-reoxygenation-induced injury in cultured bovine aortic endothelial cells (BAECs). Early passage BAECs were exposed to 24-h hypoxia followed by 4-h reoxygenation (HR). HR resulted in cell injury, as indicated by significant increases in lactate dehydrogenase (LDH) release and trypan blue stained cells (p < 0.01) and was associated with a decrease in CYP2J2 protein expression. Transfection of BAECs with the CYP2J2 cDNA resulted in increased CYP2J2 expression and arachidonic acid epoxygenase activity, compared with cells transfected with an irrelevant green fluorescent protein (GFP) cDNA. HR induced significant injury in GFP-transfected BAECs, as indicated by increases in LDH release and trypan blue-stained cells (p < 0.01); however, the HR-induced injury was markedly attenuated in CYP2J2-transfected cells (p < 0.01). HR increased cellular 8-iso-prostaglandin F2α (p < 0.05), and decreased eNOS expression, L-arginine uptake and conversion, and nitrite production (p < 0.01) in GFP-transfected BAECs. CYP2J2 transfection attenuated the HR-induced increase in 8-iso-prostaglandin F2α (p < 0.05) and decreased the amount of extracellular superoxide detected by cytochrome c reduction under normoxic conditions (p < 0.05) but did not significantly affect HR-induced decreases in eNOS expression, L-arginine uptake and conversion, and nitrite production. Treatment of BAECs with synthetic EETs and/or epoxide hydrolase inhibitors also showed protective effects against HR injury (p < 0.05). These observations suggest: (1) HR results in endothelial injury and decreased CYP2J2 expression; (2) transfection with the CYP2J2 cDNA protects against HR injury; and (3) the cytoprotective effects of CYP2J2 may be mediated, at least in part, by antioxidant effects.
• Laufs, U., & Liao, J. K. (2000). Targeting Rho in cardiovascular disease. Circulation Research, 87(Issue 7). doi:10.1161/01.res.87.7.526
• Laufs, U., Endres, M., Custodis, F., Gertz, K., Nickenig, G., Liao, J. K., & Böhm, M. (2000). Suppression of endothelial nitric oxide production after withdrawal of statin treatment is mediated by negative feedback regulation of rho GTPase gene transcription. Circulation, 102(Issue 25). doi:10.1161/01.cir.102.25.3104
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  Background - Statins improve endothelial function by upregulating endothelial nitric oxide (NO) production that is mediated by inhibiting the isoprenylation of rho GTPase. Withdrawal of statin treatment could suppress endothelial NO production and may impair vascular function. Methods and Results - To test this hypothesis, mice were treated for 14 days with 10 mg/kg atorvastatin per day; this led to the upregulation of endothelial NO synthase expression and activity by 2.3- and 3-fold, respectively. Withdrawal of statins resulted in a dramatic, 90% decrease of NO production after 2 days. In mouse aortas and cultured endothelial cells, statins upregulated the expression of rho GTPase in the cytosol, but statins blocked isoprenoid-dependent rho membrane translocation and GTP-binding activity. Inhibiting the downstream targets of rho showed that rho expression is controlled by a negative feedback mechanism mediated by the actin cytoskeleton. Measuring rho mRNA half-life and nuclear run-on assays demonstrated that statins or disruption of actin stress fibers increased rho gene transcription but not rho mRNA stability. Therefore, treatment with statins leads to the accumulation of nonisoprenylated rho in the cytosol. Withdrawing statin treatment restored the availability of isoprenoids and resulted in a massive membrane translocation and activation of rho, causing downregulation of endothelial NO production. Conclusions - Withdrawal of statin therapy in normocholesterolemic mice results in a transient increase of rho activity, causing a suppression of endothelial NO production. The underlying molecular mechanism in is a negative feedback regulation of rho gene transcription mediated by the actin cytoskeleton.
• Laufs, U., Endres, M., Stagliano, N., Amin-Hanjani, S., Chui, D. S., Yang, S. X., Simoncini, T., Yamada, M., Rabkin, E., Allen, P. G., Huang, P. L., Böhm, M., Schoen, F. J., Moskowitz, M. A., & Liao, J. K. (2000). Neuroprotection mediated by changes in the endothelial actin cytoskeleton. Journal of Clinical Investigation, 106(Issue 1). doi:10.1172/jci9639
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  Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-deficient; eNOS-/-) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion. We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion. Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 μg/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity. This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 ± 3 hours to 24 ± 4 hours. Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression. Inhibition of vascular Rho guanosine-5'-triphosphate binding activity by the 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion. No neuroprotection was observed with these agents in eNOS-/- mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.
• Marra, D. E., Simoncini, T., & Liao, J. K. (2000). Inhibition of vascular smooth muscle cell proliferation by sodium salicylate mediated by upregulation of p21(Waf1) and p27(Kip1). Circulation, 102(Issue 17). doi:10.1161/01.cir.102.17.2124
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  Background - Salicylates may have direct vascular effects by mechanisms that are independent of platelet inhibition. Methods and Results - We investigated the effect of salicylates on vascular smooth muscle cell (SMC) proliferation in response to platelet-derived growth factor (PDGF) in vitro. Salicylate concentrations of 5 and 10 mmol/L inhibited serum- or PDGF-induced SMC cell count and [3H]thymidine incorporation by 62% and 81%, respectively. There was no evidence of cellular toxicity or apoptosis as determined by trypan blue exclusion and FACS analyses. Because cell cycle progression is regulated by hyperphosphorylation of the retinoblastoma (Rb) protein, we examined the effects of salicylate on Rb hyperphosphorylation. Treatment with salicylate, but not indomethacin, inhibited nuclear factor-κB activation and completely abolished Rb hyperphosphorylation in PDGF-treated SMCs. This effect was associated with a decrease in cyclin-dependent kinase (Cdk)-2 and, to a lesser extent, Cdk-6, but not Cdk-4 activity, without changes in Cdk-2, -4, and -6 and cyclin D and E protein levels. Because Cdk-2 activity is regulated by the Cdk inhibitors p21(Waf1) and p27(Kip1), we studied the effects of salicylate on p21(Waf1) and p27(Kip1) expression. Treatment with salicylate prevented PDGF-induced downregulation of p21(Waf1) and p27(Kip1) but not of the Cdk-4/-6 inhibitor p16(Ink4). Conclusions - These findings indicate that high doses of salicylates inhibit SMC proliferation by cell cycle arrest at the G1-S phase and suggest a beneficial role for high-dose salicylates in the treatment of vascular proliferative disorders.
• Mital, S., Magneson, A., Loke, K. E., Liao, J., Forfia, P. R., & Hintze, T. H. (2000). Simvastatin acts synergistically with ACE inhibitors or amlodipine to decrease oxygen consumption in rat hearts. Journal of Cardiovascular Pharmacology, 36(Issue 2). doi:10.1097/00005344-200036001-00061
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  Statin drugs, which are cholesterol-lowering agents, can upregulate endothelial nitric oxide synthase (eNOS) in isolated endothelial cells independent of lipid lowering. We investigated the effect of short-term simvastatin administration on NO-mediated regulation of myocardial oxygen consumption (MVo2) in tissue from rat hearts. Male Wistar rats were divided into (a) control group (n = 14), and (b) simvastatin group (n = 10, 20 mg/kg/day by oral gavage). After 2 weeks, left ventricular myocardium was isolated to measure MVo2 using a Clark-type oxygen electrode, and aortic plasma nitrates and nitrites (NO(x)) were measured. Baseline plasma NO(x) levels (19 ± 2.6 in control vs. 20 ± 2.5 μ M/L in simvastatin) and baseline MVo2 (288 ± 23 in control vs. 252 ± 11 nmol/g/min; p = 0.09) were not significantly different between the two groups. NO-dependent regulation of MVo2 in response to bradykinin, ramipril, or amlodipine was augmented in simvastatin rats compared with controls (p < 0.05). Decrease of MVo2 from baseline in response to highest doses in control versus simvastatin groups was as follows-bradykinin, -28 ± 5% vs. -44 ± 6%; ramipril, -35 ± 5% vs. -50 ± 8%; and amlodipine, -32 ± 9% vs. -42 ± 3%. Response to highest dose of NO donor S-nitroso N-acetyl penicillamine (SNAP) was not significantly different in the two groups (-55 ± 5% vs. -52 ± 7%). Treatment with N(w)nitro-L-arginine methyl ester, inhibitor of NO synthesis, attenuated the effect of bradykinin, ramipril, and amlodipine on MVo2 (p < 0.05). In conclusion, short-term administration of simvastatin in rats potentiates the ability of angiotensin-converting enzyme (ACE) inhibitors and amlodipine to cause NO-mediated regulation of MVo2.
• Mital, S., Zhang, X., Zhao, G., Bernstein, R. D., Smith, C. J., Fulton, D. L., Sessa, W. C., Liao, J. K., & Hintze, T. H. (2000). Simvastatin upregulates coronary vascular endothelial nitric oxide production in conscious dogs. American Journal of Physiology - Heart and Circulatory Physiology, 279(Issue 6). doi:10.1152/ajpheart.2000.279.6.h2649
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  Statin drugs can upregulate endothelial nitric oxide (NO) synthase (eNOS) in isolated endothelial cells independent of lipid-lowering effects. We investigated the effect of short-term simvastatin administration on coronary vascular eNOS and NO production in conscious dogs and canine tissues. Mongrel dogs were instrumented under general anesthesia to measure coronary blood flow (CBF). Simvastatin (20 mg · kg-1 · day-1) was administered orally for 2 wk; afterward, resting CBF was found to be higher compared with control (P < 0.05) and veratrine-(activator of reflex cholinergic NO-dependent coronary vasodilation) and ACh-mediated coronary vasodilation were enhanced (P < 0.05). Response to endothelium-independent vasodilators, adenosine and nitroglycerin, was not potentiated. After simvastatin administration, plasma nitrate and nitrite (NO(x)) levels increased from 5.22 ± 1.2 to 7.79 ± 1.3 μM (P < 0.05); baseline and agonist-stimulated NO production in isolated coronary microvessels were augmented (P < 0.05); resting in vivo myocardial oxygen consumption (MV̇O2) decreased from 6.8 ± 0.6 to 5.9 ± 0.4 ml/min (P < 0.05); NO-dependent regulation of MV̇O2 in response to NO agonists was augmented in isolated myocardial segments (P < 0.05); and eNOS protein increased 29% and eNOS mRNA decreased 50% in aortas and coronary vascular endothelium. Short-term administration of simvastatin in dogs increases coronary endothelial NO production to enhance NO-dependent coronary vasodilation and NO-mediated regulation of MV̇O2.
• Simoncini, T., Hafezi-Moghadam, A., Brazil, D. P., Ley, K., Chin, W. W., & Llao, J. K. (2000). Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature, 407(Issue 6803). doi:10.1038/35035131
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  Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes. Controversy exists, however, concerning whether ER has a role outside the nucleus, particularly in mediating the cardiovascular protective effects of oestrogen. Here we show that the ER isoform, ERα, binds in a ligand-dependent manner to the p85α regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ERα-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ERα are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85α, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ERα with PI(3)K.
• Simoncini, T., Maffei, S., Basta, G., Barsacchi, G., Genazzani, A. R., Liao, J. K., & De Caterina, R. (2000). Estrogens and glucocorticoids inhibit endothelial vascular cell adhesion molecule-1 expression by different transcriptional mechanisms. Circulation Research, 87(Issue 1). doi:10.1161/01.res.87.1.19
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  The antiatherogenic effect of estrogen is mediated, in part, by inhibitory effects on endothelial vascular cell adhesion molecule-1 (VCAM-1) expression. To determine the mechanism by which estrogen regulates VCAM-1 expression, we compared the effect of 17β-estradiol (E2) and of the glucocorticoid dexamethasone (Dex) on lipopolysaccharide (LPS)-induced VCAM-1 expression in human endothelial cells. E2 decreased LPS-induced VCAM-1 mRNA and protein expression to a greater extent than Dex. Dex, but not E2, stabilized VCAM-1 mRNA. This correlated with inhibition of monocytoid U937 cell adhesion to endothelial cells. Transfection of endothelial cells with a functional VCAM-1 promoter construct showed that E2 inhibited LPS-induced VCAM-1 gene transcription more potently than did Dex. However, using a truncated construct containing only the nuclear factor-κB (NF- κB)-responsive elements but lacking the consensus sequences for activator protein-1 (AP-1) and GATA, E2 and Dex had similar inhibitory effects. Consistently, gel-shift assays showed that E2 and Dex comparably inhibit LPS-induced activation of NF-κB, whereas E2 inhibited LPS-induced activation of AP-1 and GATA to a greater extent than Dex. E2 inhibition of NF-κB after LPS treatment was associated with decreased inhibitor κB (IκB) kinase activity and with a stabilization of the NF-κB inhibitor IκBα. These results indicate that E2 decreases VCAM-1 gene expression through the inhibition of NF-κB, AP-1, and GATA and suggest novel mechanisms for the antiatherogenic effect of estrogen on the vascular wall.
• Spiecker, M., Darius, H., & Liao, J. K. (2000). A functional role of IκB-ε in endothelial cell activation. Journal of Immunology, 164(Issue 6). doi:10.4049/jimmunol.164.6.3316
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  The NF-κB inhibitor IκB-ε is a new member of the IκB protein family, but its functional role in regulating NF-κB-mediated induction of adhesion molecule expression is unknown. In vascular endothelial cells, IκB-ε associates predominantly with the NF-κB subunit Rel A and to a lesser extent with c-Rel, whereas IκB-α and IκB-β associate with Rel A only. Following stimulation with TNF-α, pyrrolidine dithiocarbamate (PDTC), N- acetylcysteine, and dexamethasone prevented IκB kinase-induced IκB-α, but not IκB-β or IκB-ε phosphorylation and degradation. Since the activation of NF-κB is required for the induction of adhesion molecule expression, we examined the role of IκB-ε in the transactivation of promoters from VCAM-1, ICAM-1, and E-selectin. Using reporter gene constructs of adhesion molecule promoters, PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 promoter activity. Subcloning of κB cis-acting elements of VCAM-1, E- selectin, and ICAM-1 into a heterologous promoter construct revealed that PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 κB promoter activity. By electrophoretic mobility shift assay, NF-κB heterodimers containing c-Rel specifically bind to the κB motif in the ICAM- 1, but not VCAM-1 or E-selectin promoter. Indeed, overexpression of c-Rel induced ICAM-1 κB promoter activity to a greater extent than that of E- selectin and overexpression of IκB-ε inhibited ICAM-1 and VCAM-1 promoter activity in endothelial cells. These findings indicate that c-Rel-associated IκB-ε is involved in the induction of ICAM-1 expression.
• Yamada, M., Huang, Z., Dalkara, T., Endres, M., Laufs, U., Waeber, C., Huang, P. L., Liao, J. K., & Moskowitz, M. A. (2000). Endothelial nitric oxide synthase-dependent cerebral blood flow augmentation by L-arginine after chronic statin treatment. Journal of Cerebral Blood Flow and Metabolism, 20(Issue 4). doi:10.1097/00004647-200004000-00008
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  Nitric oxide, a product of nitric oxide synthase activity, relaxes vascular smooth muscle and elevates brain blood flow. We evaluated the importance of eNOS to cerebral blood flow augmentation after L-arginine infusion and increases in flow after eNOS upregulation in SV-129 mice. Blood flow was measured by laser-Doppler flowmetry before and after L-arginine infusion (450 mg/kg during a 15-minute period) or measured by 14C- iodoamphetamine indicator fractionation or 14C-iodoantipyrine tissue equilibration techniques. rCBF increased by 26% (laser Doppler flowmetry) after L-arginine infusion but did not change in mutant mice deficient in eNOS expression. After eNOS upregulation by chronic simvastatin treatment (2 mg/kg subcutaneously, daily for 14 days), L-arginine amplified and sustained the hyperemia (38%) and increased absolute brain blood flow from 86 ± 7 to 119 ± 10 mL/100 g per minute. Furthermore, pretreatment with simvastatin enhanced blood flow within ischemic brain tissue after middle cerebral artery occlusion. Together, these findings suggest that eNOS activity is critical for blood flow augmentation during acute L-arginine infusion, and chronic eNOS upregulation combined with L-arginine administration provides a novel strategy to elevate cerebral blood flow in the normal and ischemic brain.
• Laufs, U., Endres, M., & Liao, J. K. (1999). Regulation of endothelial NO production by Rho GTPase. Medizinische Klinik, 94(Issue 4). doi:10.1007/bf03044857
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  Endothelial-derived nitric oxide (NO) is an important mediator of vascular function. Clinical studies indicate that HMG-CoA reductase inhibitors (statins) improve endothelial function and reduce the incidence of stroke and myocardial infarction. Treatment of human endothelial cells with statins increased the expression of endothelial NO synthase (eNOS) protein and mRNA expression. Statins increased eNOS mRNA half-life but did not change eNOS gene transcription. Inhibition of mevalonate synthesis by statins not only blocks the formation of cholesterol but also of isoprenoids. The upregulation of eNOS expression by statins was independent of cholesterol but mediated via the inhibition of the isoprenoid geranylgeraniol whereas farnesiol had no effect one eNOS. Immunoblot analyses, (35S)-GTPγS- binding assay and transfections studies revealed that statins upregulated eNOS expression by blocking the geranylgeranylation of the GTPase Rho which is necessary for its membrane-associated activity. Studies with mice showed, that statin treatment upregulates eNOS expression and function independent of serum cholesterol levels. Prophylactic treatment with statins augmented cerebral blood flow and reduced cerebral infarcts in normocholesterolemic mice. These effects of statins were completely absent in eNOS-deficit mice indicating that enhanced eNOS activity by statins is the predominant mechanism by which these agents protect against cerebral injury. Our results suggest that statins provide a novel prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia. Upregulation of eNOS by inhibiting Rho may provide a new pharmacologic target for the treatment of arteriosclerosis, pulmonary hypertension, and heart failure.
• Laufs, U., Marra, D., Node, K., & Liao, J. K. (1999). 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing Rho GTPase-induced down-regulation of p27(Kip1). Journal of Biological Chemistry, 274(Issue 31). doi:10.1074/jbc.274.31.21926
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  The mechanism by which platelet-derived growth factor (PDGF) regulates vascular smooth muscle cell (SMC) DNA synthesis is unknown, but may involve isoprenoid intermediates of the cholesterol biosynthetic pathway. Inhibition of isoprenoid synthesis with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, simvastatin (Sim, 1-10 μM), inhibited PDGF-induced SMC DNA synthesis by >95%, retinoblastoma gene product hyperphosphorylation by 90%, and cyclin-dependent kinases (cdk)-2, -4, and -6 activity by 80 ± 5, 50 ± 3, and 48 ± 3%, respectively. This correlated with a 20-fold increase in p27(Kip1) without changes in p16, p21(waf1), or p53 levels compared with PDGF alone. Since Ras and Rho require isoprenoid modification for membrane localization and are implicated in cell cycle regulation, we investigated the effects of Sim on Ras and Rho. Up-regulation of p27(Kip1) and inhibition of Rho but not Ras membrane translocation by Sire were reversed by geranylgeranylpyrophosphate, but not farnesylpyrophosphate. Indeed, inhibition of Rho by Clostridium botulinum C3 transferase or overexpression of dominant-negative N19RhoA mutant increased p27(Kip1) and inhibited retinoblastoma hyperphosphorylation. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 decreased p27(Kip1) and increased SMC DNA synthesis. These findings indicate that the down-regulation of p27(Kip1) by Rho GTPase mediates PDGF-induced SMC DNA synthesis and suggest a novel direct effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors on the vascular wall.
• Lu, M., Perez, V. L., Ma, N., Miyamoto, K., Peng, H. B., Liao, J. K., & Adamis, A. P. (1999). VEGF increases retinal vascular ICAM-1 expression in vivo. Investigative Ophthalmology and Visual Science, 40(Issue 8).
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  Purpose. Intraocular injections of vascular endothelial growth factor (VEGF), a peptide implicated in the pathogenesis of diabetic retinopathy, can induce retinal ischemia. Diabetic retinal ischemia may be caused, in part, by the adhesion of leukocytes to the retinal vasculature. In this study, the ability of VEGF to increase the expression of intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules in capillary endothelium and the retinal vasculature was examined. Methods. The expression of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin on human brain capillary endothelial cell monolayers exposed to VEGF was quantitated by immunoassay. The effect of VEGF on retinal vascular ICAM-1 expression was determined in ICAM-1 immunofluorescence studies of retinal flat-mounts and in RNase protection assays. Results. VEGF increased capillary endothelial cell ICAM-1 levels in a dose- and time-dependent manner (6 -24 hours, plateau after 6 hours; EC50, 25 ng/ml). VEGF failed to alter E- selectin, P-selectin, or VCAM-1 levels under the conditions tested. Intravitreal injections of pathophysiologically relevant concentrations of VEGF increased ICAM-1 protein and mRNA levels in the retinal vasculature. Conclusions. VEGF increases retinal vascular ICAM-1 expression. VEGF-induced increases in ICAM-1 may promote retinal leukostasis in diabetic eyes.
• Miyamoto, A., Yang, S. X., Laufs, U., Ruan, X. L., & Liao, J. K. (1999). Activation of guanine nucleotide-binding proteins and induction of endothelial tissue-type plasminogen activator gene transcription by alcohol. Journal of Biological Chemistry, 274(Issue 17). doi:10.1074/jbc.274.17.12055
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  The mechanism by which moderate alcohol ingestion lowers the risk of cardiovascular disease is unknown but may be due, in part, to the ability of alcohol to increase the level of tissue-type plasminogen activator (t-PA). Human endothelial cells were treated with low concentrations, of ethanol (0.25-25 mM, 0-24 h), which are associated with moderate alcohol consumption. Although treatment with ethanol alone did not affect t-PA gene transcription or mRNA expression, it augmented isoproterenol (ISO)-stimulated t-PA gene transcription and mRNA levels by 3.4- and 2.8-fold, respectively, and decreased plasminogen activator inhibitor-1 mRNA levels by 65%. These effects of ethanol correlated with 2.5 and 6.9-fold increases in ISO-stimulated cyclic AMP levels and 4x-cyclic AMP response element heterologous promoter activity, respectively. To determine whether alcohol-induced changes in agonist-stimulated cyclic AMP levels were because of modulation of guanine nucleotide-binding proteins (G proteins), we assessed the effects of ethanol on Gα(s) and Gα(i2). Although ethanol did not affect the expression of Gα(s) or Gα(i2), it increased ISO-stimulated Gα(s) GTPase and GTP binding activity by 2.2- and 2.9-fold and decreased UK14304-stimulated Gα(i2) GTPase and GTP binding activity by 38 and 80%. These results indicate that treatment with relatively low concentrations of ethanol enhances agonist-stimulated cyclic AMP-dependent t-PA gene transcription in vascular endothelial cells through differential modulation of G protein.
• Node, K., Huo, Y., Ruan, X., Yang, B., Spiecker, M., Ley, K., Zeldin, D. C., & Liao, J. K. (1999). Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science, 285(Issue 5431). doi:10.1126/science.285.5431.1276
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  The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenases that have vasodilatory properties similar to that of endothelium-derived hyperpolarizing factor. The cytochrome P450 isoform CYP2J2 was cloned and identified as a potential source of EETs in human endothelial cells. Physiological concentrations of EETs or overexpression of CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression, and EETs prevented leukocyte adhesion to the vascular wall by a mechanism involving inhibition of transcription factor NF-κB and IκB kinase. The inhibitory effects of EETs were independent of their membrane- hyperpolarizing effects, suggesting that these molecules play an important nonvasodilatory role in vascular inflammation.
• Rajavashisth, T. B., Liao, J. K., Galis, Z. S., Tripathi, S., Laufs, U., Tripathi, J., Chai, N. N., Xu, X. P., Jovinge, S., Shah, P. K., & Libby, P. (1999). Inflammatory cytokines and oxidized low density lipoproteins increase endothelial cell expression of membrane type 1-matrix metalloproteinase. Journal of Biological Chemistry, 274(Issue 17). doi:10.1074/jbc.274.17.11924
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  We investigated whether inflammatory cytokines or oxidized low density lipoproteins (Ox-LDL) present in human atheroma modulate extracellular matrix degradation by inducing membrane type 1-matrix metalloproteinase (MT1-MMP) expression. Cultured human endothelial cells (EC) constitutively expressed MT1-MMP mRNA and protein with enzymatic activity. Tumor necrosis factor-α (TNF-α), interleukin-1α, or interleukin-1β caused a time-dependent increase in the steady-state MT1-MMP mRNA levels within 4 h of exposure, peaking about 4-fold by 6 h, and remaining elevated for 12 h. Increased MT1- MMP mRNA correlated with a 2.5-fold increase in MT1-MMP protein in EC membranes. Ox-LDL also increased MT1-MMP mRNA levels that varied with the duration of exposure and degree of LDL oxidation. The increase in MT1-MMP mRNA occurred within 6 h of exposure to Ox-LDL and peaked over 3-fold by 6 h. Ox-LDL, but not native LDL, increased MT1-MMP protein by 2-fold in EC membranes. A combination of TNF-α and Ox-LDL was additive in increasing MT1- MMP expression. Nuclear run-on assays showed that TNF-α or Ox-LDL augmented steady-state mRNA levels by increased transcription of the MT1-MMP gene. These findings indicate that activation of EC by inflammatory cytokines and/or Ox-LDL increase MT1-MMP expression. Since MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokines or Ox-LDL may influence extracellular matrix remodeling.
• Spiecker, M., & Liao, J. K. (1999). Assessing induction of IκB by nitric oxide. Methods in Enzymology. doi:10.1016/s0076-6879(99)00142-1
• De Caterina, R., Bernini, W., Carluccio, M. A., Liao, J. K., & Libby, P. (1998). Structural requirements for inhibition of cytokine-induced endothelial activation by unsaturated fatty acids. Journal of Lipid Research, 39(Issue 5). doi:10.1016/s0022-2275(20)33875-x
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  Dietary long-chain fatty acids (FA) may influence pathological processes involving endothelial activation, including inflammation and atherosclerosis. We have previously shown that the n-3 FA docosahexaenoate (DHA) inhibits endothelial activation in the range of nutritionally achievable plasma concentrations. The present study assessed structural determinants for this effect. Saturated, monounsaturated, and n-6 and n-3 polyunsaturated FA were incubated with cultured endothelial cells for 24-72 h alone, and then in the presence of interleukin-1, tumor necrosis factor, or bacterial lipopolysaccharide for an additional 24 h before assessing the expression of the vascular cell adhesion molecule-1 (VCAM-1) or other products of endothelial activation. No FA tested per se elicited endothelial activation. While saturated FA did not inhibit cytokine-induced expression of adhesion molecules, a progressively increasing inhibitory activity was observed, for the same chain length, with an increase in double bonds. Comparison of FA with the same length and number of unsaturation and only differing for the double bond position or for the cis/trans configuration indicated no difference in inhibitory potency, indicating no effect of the double bond position or configuration. As judged by Northern analysis, these latter FA also inhibited VCAM-1 messenger RNA steady state levels to the same extent, indicating a pre-translational site of action attributable to the single double bond. Thus the double bond is the minimum necessary and sufficient requirement for FA inhibition of endothelial activation. These properties are likely relevant to the anti-atherogenic and anti-inflammatory properties ascribed to n-3 FA, which are able to accommodate the highest number of double bonds in a fatty acid of given chain length.
• Endres, M., Laufs, U., Huang, Z., Nakamura, T., Huang, P., Moskowitz, M. A., & Liao, J. K. (1998). Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proceedings of the National Academy of Sciences of the United States of America, 95(Issue 15). doi:10.1073/pnas.95.15.8880
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  The treatment of ischemic strokes is limited to prophylactic agents that block the coagulation cascade. Here, we show that cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, protect against cerebral injury by a previously unidentified mechanism involving the selective up-regulation of endothelial NO synthase (eNOS). Prophylactic treatment with HMG-CoA reductase inhibitors augments cerebral blood flow, reduces cerebral infarct size, and improves neurological function in normocholesterolemic mice. The up-regulation of eNOS by HMG-CoA reductase inhibitors is not associated with changes in serum cholesterol levels, but is reversed by cotreatment with L-mevalonate and by the downstream isoprenoid, geranylgeranyl pyrophosphate and not by farnesyl pyrophosphate. The blood flow and neuroprotective effects of HMG-CoA reductase inhibitors are completely absent in eNOS-deficient mice, indicating that enhanced eNOS activity by HMG-CoA reductase inhibitors is the predominant if not the only mechanism by which these agents protect against cerebral injury. Our results suggest that HMG-CoA reductase inhibitors provide a prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia.
• Laufs, U., & Liao, J. K. (1998). Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. Journal of Biological Chemistry, 273(Issue 37). doi:10.1074/jbc.273.37.24266
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  The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 μM), in the presence of L-mevalonate (200 μM), geranylgeranylpyrophosphate (GGPP, 1-10 μM), farnesylpyrophosphate (FPP, 5- 10 μM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 ± 15% and 180 ± 11%, respectively. Co- treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPγS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 ± 5% and 78 ± 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 μg/ ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.
• Laufs, U., La Fata, V., Plutzky, J., & Liao, J. K. (1998). Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation, 97(Issue 12). doi:10.1161/01.cir.97.12.1129
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  Background - Oxidized low-density lipoprotein (ox-LDL) causes endothelial dysfunction in part by decreasing the availability of endothelial nitric oxide (NO). Although HMG CoA reductase inhibitors restore endothelial function by reducing serum cholesterol levels, it is not known whether they can also directly upregulate endothelial NO synthase (ecNOS) activity. Methods and Results - Human saphenous vein endothelial cells were treated with ox-LDL (50 μg/mL thiobarbituric acid reactive substances 12 to 16 nmol/mg) in the presence of HMG CoA reductase inhibitors simvastatin and lovastatin. In a time-dependent manner, ox-LDL decreased ecNOS mRNA and protein levels (91±4% and 67±8% reduction after 72 hours, respectively). Both simvastatin (1 μmol/L) and lovastatin (10 μmol/L) upregulated ecNOS expression by 3.8-fold and 3.6-fold, respectively, and completely prevented its downregulation by ox-LDL. These effects of simvastatin on ecNOS expression correlated with changes in ecNOS activity. Although L-mevalonate alone did not affect ecNOS expression, cotreatment with L-mevalonate completely reversed ecNOS upregulation by simvastatin. Actinomycin D studios revealed that simvastatin stabilized ecNOS mRNA (τ( 1/4 ), 43 versus 35 hours). Nuclear run-on assays and transient transfection studies with a -1.6 kb ecNOS promoter construct showed that simvastatin did not affect ecNOS gene transcription. Conclusions - Inhibition of endothelial HMG CoA reductase upregulates ecNOS expression predominantly by posttranscriptional mechanisms. These findings suggest that HMG CoA reductase inhibitors may have beneficial effects in atherosclerosis beyond that attributed to the lowering of serum cholesterol by increasing ecNOS activity.
• Peng, H. B., Spiecker, M., & Liao, J. K. (1998). Inducible nitric oxide: An autoregulatory feedback inhibitor of vascular inflammation. Journal of Immunology, 161(Issue 4). doi:10.4049/jimmunol.161.4.1970
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  Inducible nitric oxide (iNO) is produced at sites of vascular inflammation by resident and nonresident vascular wall cells, but its role in the inflammatory process is not known. In this study, we show that a novel function of iNO is to terminate inflammatory processes. We find that iNO produced by murine macrophage-like cells, RAW264.7, can inhibit cytokine- induced endothelial cell activation in a separated and mixed endothelial- RAW264.7 coculture system. Both iNO production and endothelial VCAM-1 expression were induced simultaneously with bacterial LPS and murine-specific IFN-γ. Inhibition of iNO synthase (iNOS) activity with Nω-monomethyl-L- arginine in endothelial-RAW264.7 cocultures, stimulated with murine-specific IFN-γ and LPS, decreased iNO production by 86%, augmented VCAM-1 and iNOS expression in endothelial and RAW264.7 cells, respectively, and increased monocyte adhesion to the endothelial cell surface. Transient transfection studies using various VCAM-1 promoter constructs demonstrated that inhibitory effects of iNO on VCAM-1 gene transcription were mediated, in part, by inhibitory effects of iNO on κB cis-acting elements. Immunofluorescence studies using an Ab to the RelA (p65) subunit of nuclear factor-κB revealed that iNO inhibited the activation of nuclear factor-κB. These studies indicate that iNO attenuates iNOS expression in macrophages and inhibits monocyte adhesion to endothelial cells, and suggest that endogenously derived iNO may be an important autoregulatory inhibitor of vascular inflammation.
• Spiecker, M., Darius, H., Kaboth, K., Hübner, F., & Liao, J. K. (1998). Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants. Journal of Leukocyte Biology, 63(Issue 6). doi:10.1002/jlb.63.6.732
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  Although nitric oxide (NO) and antioxidants inhibit adhesion molecule expression, their inhibitory effects on nuclear factor κB (NF-κB) activation may differ. The NO donors, but not 8-bromo-cGMP, decreased tumor necrosis factor α (TNF-α)-induced VCAM-1, ICAM-1, and E-selectin expression by 11-70%. In contrast, NAC completely abolished VCAM-1 and E-selectin expression and decreased ICAM-1 expression by 56%. Gel shift assays demonstrate that NF-κB activation was inhibited by both NO and antioxidants. The activation of NF-κB involves the phosphorylation and degradation of its cytoplasmic inhibitor IκB-α by 26S proteasomes. The 26S proteasome inhibitor MG132 prevented the degradation of phosphorylated IκB-α. NAC inhibited IκB kinase (IKK) activity and prevented IKB-α phosphorylation and degradation. In contrast, NO did not inhibit IKK activity, IκB-α phosphorylation, or IκB-α degradation. However, NO, but not antioxidants, induced IκB-α promoter activity. The inhibitory effects of NO on adhesion molecule expression, therefore, differs from that of antioxidants in terms of the mechanism by which NF-κB is inactivated.
• Hong, Y. H., Peng, H. B., Fata, V. L., & Liao, J. K. (1997). Hydrogen peroxide-mediated transcriptional induction of macrophage colony-stimulating factor by TGF-β1. Journal of Immunology, 159(Issue 5).
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  TGF-β1 and macrophages are important regulators of tissue fibrosis and remodeling. Here we show that TGF-β1 induces the expression of macrophage-CSF (M-CSF) in vascular endothelial cells via a signaling pathway(s) involving hydrogen peroxide (H 2O 2). In a time-dependent manner, TGF-β1 produced a 10- and a 6-fold increase in M-CSF mRNA and protein levels after 12 h, respectively. This increase in M-CSF expression was attenuated by a nitric oxide donor, S-nitrosoglutathione (GSNO), and by a nonspecific oxidase inhibitor, diphenylene iodonium. Furthermore, the TGF-β1-induced M-CSF mRNA expression was inhibited by catalase, but not by superoxide dismutase, suggesting that H 2O 2 rather than superoxide anion (O 2 -) is the primary mediator of the effects of TGF-β1. Transient transfection studies using deletional M-CSF promoter constructs demonstrated that TGF-β1 produced a 13-fold induction in M-CSF promoter activity that was repressed by >85% with GSNO and catalase, in part through inhibitory effects on κB cis-acting elements. Electrophoretic mobility shift assays revealed that the activation of nuclear factor-κB by TGF-β1 was also inhibited by GSNO and catalase, but not by superoxide dismutase. In a concentration-dependent manner, treatment with exogenous H 2O 2 produced 14- and 4.6-fold increases in M-CSF promoter activity and mRNA expression, respectively. These results indicate that the generation of H 2O 2 and activation of NF-κB by TGF-β1 are required for the induction of M-CSF gene transcription.
• Laufs, U., La Fata, V., & Liao, J. K. (1997). Inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase blocks hypoxia-mediated down-regulation of endothelial nitric oxide synthase. Journal of Biological Chemistry, 272(Issue 50). doi:10.1074/jbc.272.50.31725
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  Hypoxia induces vasoconstriction, in part, by down-regulating endothelial cell nitric oxide synthase (ecNOS) expression. Previous studies indicate that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors improve endothelium-dependent relaxation by increasing of ecNOS activity. To determine whether HMG CoA reductase inhibitors can prevent hypoxia-mediated down-regulation of ecNOS function and expression, human endothelial cells were exposed to hypoxia (3% O2) in the presence of HMG CoA reductase inhibitors simvastatin and lovastatin for various durations (0-48 h). Hypoxia decreased ecNOS protein and mRNA levels in a time-dependent manner, resulting in a 4- and 9-fold reduction after 48 h, respectively. In a concentration-dependent manner, simvastatin, and to a lesser extent, lovastatin, prevented the down-regulation of ecNOS expression by hypoxia. Simvastatin-induced changes in ecNOS expression correlated with changes in endothelial NO production and were reversed by treatment with L-mevalonate. Actinomycin D studies revealed that under hypoxic conditions, simvastatin increased ecNOS mRNA half-life from 13 to 38 h. Nuclear run-on studies showed that simvastatin had no effect on repression of ecNOS gene transcription by hypoxia. These results indicate that HMG CoA reductase inhibitors regulate ecNOS function and expression through changes in ecNOS mRNA stability and suggest that treatment with HMG CoA reductase inhibitors may have beneficial effects in patients with hypoxia-mediated pulmonary hypertension.
• Miyamoto, A., Laufs, U., Pardo, C., & Liao, J. K. (1997). Modulation of bradykinin receptor ligand binding affinity and its coupled G-proteins by nitric oxide. Journal of Biological Chemistry, 272(Issue 31). doi:10.1074/jbc.272.31.19601
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  To determine whether nitric oxide (NO) can modulate bradykinin (BK) signaling pathways, we treated endothelial cells with an NO donor, S- nitrosoglutathione (GSNO), to determine its effect(s) on G-proteins (G(i) and G(q)) that are coupled to the type II kinin (BK2) receptor. Radioligand binding assays and Western analyses showed that GSNO (10-500 μM, 0-72 h) did not alter the expression of BK2 receptor, G(i), or G(q). However, GSNO cause a 6-fold increase in basal cGMP production and decreased high affinity BK bindings sites and GTPase activity by 74 and 85%, respectively. The cGMP analogue, dibutyryl-cGMP, also inhibited BK-stimulated GTPase activity by 74% suggesting that some of the effects of NO may be mediated through activation of guanylyl cyclase. The NO synthase inhibitor, N(ω)-monomethyl-L-arginine, inhibited endogenous NO synthase activity and cGMP production by 91 and 76%, respectively, but increased BK-stimulated GTPase activity by 61%. To determine which G-proteins are affected by NO, we performed GTP binding assays with [35S]GTPγS followed by immunoprecipitation with specific G- protein antisera. Both GSNO and dibutyryl-cGMP increased basal G-protein GTP binding activities by 18-26%. However, GSNO decreased BK-stimulated Gα(i2), Gα(i3), and Gα(q/11) GTP binding activity by 93, 61, and 90%, respectively, whereas epinephrine-stimulated Gα(s) GTP binding activity was unaffected. These results suggest that NO can modulate BK signaling pathways by selectively inhibiting G-proteins of the G(i) and G(q) family.
• Spiecker, M., Peng, H. B., & Liao, J. K. (1997). Inhibition of endothelial vascular cell adhesion molecule-1 expression by nitric oxide involves the induction and nuclear translocation of IκBα. Journal of Biological Chemistry, 272(Issue 49). doi:10.1074/jbc.272.49.30969
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  The induction of vascular cell adhesion molecule-1 (VCAM-1) expression by tumor necrosis factor (TNF)-α requires the activation of nuclear factor- κB (NF-κB) via a process involving the phosphorylation and degradation of its cytoplasmic inhibitor, IκBα. We have shown that nitric oxide (NO) decreases VCAM-1 expression via inhibition of NF-κB activation. To determine how NO inhibits NF-κB, we studied the fate of IκBα following TNF-α stimulation in the presence of NO donors S-nitrosoglutathione and sodium nitroprusside. Activation of NF-κB by TNF-α occurred within 15 min and coincided with rapid degradation of IκBα. Co-treatment with NO donors did not prevent IκBα phosphorylation or degradation. However, after 2 h of TNF- α stimulation, NO donors inhibited NF-κB activation and augmented IκBα resynthesis and nuclear translocation by 2.5- and 3-fold, respectively. This correlated with a 75% reduction in TNF-α-induced VCAM-1 expression. In a time-dependent manner, NO donors alone caused the nuclear translocation of IκBα. To confirm that NO donors have similar effects as endogenously derived NO, murine macrophage-like cells, RAW264.7, were co-cultured with endothelial cells. Induction of RAW264.7-derived NO inhibited lipopolysaccharide-induced endothelial VCAM-1 expression, which was reversed by the NO synthase inhibitor N(ω)-monomethyl-L-arginine. These finding, indicate that NO inhibits NF-κB activation and VCAM-1 expression by increasing the expression and nuclear translocation of IκBα.
• Shin, W. S., Hong, Y. H., Peng, H. B., De Caterina, R., Libby, P., & Liao, J. K. (1996). Nitric oxide attenuates vascular smooth muscle cell activation by interferon-γ: The role of constitutive NF-κb activity. Journal of Biological Chemistry, 271(Issue 19). doi:10.1074/jbc.271.19.11317
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  Atherogenesis involves cellular immune responses and altered vascular smooth muscle cell (SMC) function. Cytokines such as interleukin (IL)-1α1 and interferon-γ (IFN-γ) may contribute to this process by activating SMC. To determine whether the anti-atherogenic mediator, nitric oxide (̇NO), can modulate cytokine-induced SMC activation, we investigated the effects of various ̇NO-generating compounds on the expression of intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). Induction of ICAM-1 expression by IL-1α and VCAM-1 expression by IFN-γ was attenuated by ̇NO donors but not by cGMP analogues. Nuclear run-on assays and transfection studies using various VCAM-1 promoter constructs linked to the chloramphenicol acetyltransferase reporter gene showed that ̇NO repressed IFN-γ-induced VCAM-1 gene transcription, in part, through inhibition of nuclear factor-κ B (NF-κB). Electrophoretic mobility shift assay revealed that SMC possess basal constitutive NF-κB activity, which was augmented by treatment with IL-1α. In contrast, IFN-γ induced and activated Interferon regulatory factor (IRF)-1 but had little effect on basal constitutive NF-κB activity. ̇NO donors had no inhibitory effect on IRF-1 activation but did inhibit basal and IL-α-stimulated NF-κB activation. These findings suggest that the induction of ICAM-1 and VCAM-1 expression requires NF-κB activation and that ̇NO attenuates IFN-γ-induced VCAM-1 expression primarily by inhibiting basal constitutive NF-κB activity in SMC.
• Shin, W. S., Kawaguchi, H., Liao, J. K., Wang, Y., Inukai, M., & Toyo-Oka, T. (1996). Toxic action of nitric oxide on myocardial cells: Direct evidence from gene transfer in vivo. Journal of Cardiac Failure, 2(Issue 1). doi:10.1016/s1071-9164(96)80071-6
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  To examine whether nitric oxide (NO) has a protective effect against Ca2+overdose or a beneficial action on myocardial cells, we employed direct gene-transfer of endothelial (type III) nitric oxide synthase (eNOS), using HVJ (Sendai virus) coated liposomes and beta-galactosidase (lac-z) as a marker for the transfection. The transfection efficiency of the lac-z gene was comparable with adenovirus as a vector, though the subsequent inflammation was much improved. The lac-z gene transfection was restricted to myoplasm between two intercalated discs, indicating that the transfected gene does not permeate the disc. Co-transfection with human eNOS gene revealed degraded myoplasm of not only transfected cells but adjacent myocytes, fibrotic changes and infiltration of mononuclear cells seven days after the transfection. Electron microscopy of the lesions revealed a huge accumulation of mitochondria and loss of myofilaments, though fragmentation of nucleus or cytoplasm was not obvious. We conclude that an expression of human eNOS gene in cardiomyocytes causes a degenerative process, incompatible with typical apoptosis.
• De Caterina, R., Libby, P., Peng, H. B., Thannickal, V. J., Rajavashisth, T. B., Gimbrone, M. A., Shin, W. S., & Liao, J. K. (1995). Nitric oxide decreases cytokine-induced endothelial activation: Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. Journal of Clinical Investigation, 96(Issue 1). doi:10.1172/jci118074
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  To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1α-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1α, IL-1β, IL-4, tumor necrosis factor (TNFα), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-I, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF-κB. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall.
• Liao, J. K., & Clark, S. L. (1995). Regulation of G-protein αi2 subunit expression by oxidized low-density lipoprotein. Journal of Clinical Investigation, 95(Issue 4).
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  Oxidized low-density lipoprotein (LDL) inhibits signalling pathways mediated by pertussis toxin-sensitive guanine nucleotide-binding proteins (Gi proteins). To determine whether this inhibition is due to altered G protein αi subunit expression, mRNA and protein levels of αi isoforms were assessed in bovine aortic endothelial cells treated with oxidized LDL (0-100 μg/ml, 0-72 h). Oxidized LDL did not affect the expression of αi3, but did cause time- and concentration-dependent decrease in αi2 mRNA and protein resulting in a 3.2- and 3.5-fold reduction, respectively, after 72 h. This decrease in αi2 coincided with a 86% decrease in αi2 GTPase activity. Nuclear run-off studies did not show any significant effect of oxidized LDL on αi2 or αi3 transcription. In the presence of actinomycin D, oxidized LDL shortened the t1/2 of αi2 mRNA from 16 h to 8 h which was attenuated by cycloheximide. In addition, pulse-chase labelling with [35S]methionine revealed that oxidized LDL reduced the t1/2 of αi2 protein from 27 to 14 h. Our results indicate that oxidized LDL can modulate receptor-Gi coupling by downregulating the expression of αi2, but not αi3. The mechanism involves both mRNA destabilization and protein degradation.
• Liao, J. K., Shin, W. S., Lee, W. Y., & Clark, S. L. (1995). Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. Journal of Biological Chemistry, 270(Issue 1). doi:10.1074/jbc.270.1.319
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  The atherogenic effects of low-density lipoprotein (LDL) may be mediated, in part, by its effect(s) on endothelial-derived nitric oxide (NO). To determine whether LDL can modulate NO production by changing NO synthase expression, we treated human saphenous vein endothelial cells with increasing concentrations of native or oxidized LDL (0-100 μg/ml) for various durations (0-72 h). Oxidized, but not native LDL caused a time-dependent decrease in steady-state NO synthase mRNA levels. This coincided with a maximal 56% decrease in NOS activity as determined by [3H]arginine to [3H]citrulline conversion. In the presence of actinomycin D, treatment with oxidized LDL reduced the half-life of NO synthase mRNA from 36 to 10 h. This decrease in NO synthase mRNA correlated with the degree of LDL oxidation and was attenuated by pretreatment with cycloheximide. Nuclear run-off studies showed a biphasic transcriptional pattern of NO synthase gene with an initial 25% decrease during the first 6 h followed by a maximal 2.2-fold increase over baseline during the subsequent 18 h. These results indicate that oxidized LDL regulates endothelial NOS expression through a combination of early transcriptional inhibition and post-transcriptional mRNA destabilization.
• Liao, J. K., Zulueta, J. J., Yu, F. S., Peng, H. B., Cote, C. G., & Hassoun, P. M. (1995). Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. Journal of Clinical Investigation, 96(Issue 6). doi:10.1172/jci118332
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  Oxygen (O2) may regulate pulmonary vascular resistance through changes in endothelial nitric oxide (NO) production. To determine whether constitutive NO synthase (cNOS) is regulated by O2, we assessed cNOS expression and activity in bovine pulmonary artery endothelial cells exposed to different concentrations of O2. In a time-dependent manner, changes in O2 concentration from 95 to 3% produced a progressive decrease in cNOS mRNA and protein levels resulting in 4.8- and 4.3-fold reductions after 24 h, respectively. This correlated with changes in cNOS activity as determined by nitrite measurements. Compared with 20% O2, cNOS activity was increased 1.5- fold in 95% O2 and decreased 1.9-fold in 3% O2. A decrease in O2 concentration from 95 to 3% shortened cNOS mRNA half-life from 46 to 24 h and caused a 20-fold repression of cNOS gene transcription. Treatment with cycloheximide produced a threefold increase in cNOS mRNA at all O2 concentrations, suggesting that cNOS mRNA expression is negatively regulated under basal condition. We conclude that O2 upregulates cNOS expression through transcriptional and posttranscriptional mechanisms. A decrease in cNOS activity in the presence of low O2 levels, therefore, may contribute to hypoxia-induced vasoconstriction in the pulmonary circulation.
• Peng, H. B., Libby, P., & Liao, J. K. (1995). Induction and stabilization of IκBα by nitric oxide mediates inhibition of NF-κB. Journal of Biological Chemistry, 270(Issue 23). doi:10.1074/jbc.270.23.14214
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  To determine the mechanism(s) by which the endogenous mediator nitric oxide (NO) inhibits the activation of transcription factor NF-κB, we stimulated human vascular endothelial cells with tumor necrosis factor-α in the presence of two NO donors, sodium nitroprusside and S-nitrosoglutathione. Electrophoretic mobility shift assays demonstrated that both NO donors inhibited NF-κB activation by tumor necrosis factor-α. This effect was not mediated by guanylyl cyclase activation since the cGMP analogue 8-bromo-cGMP had no similar effect. Inhibition of endogenous constitutive NO production by L-N-monomethylarginine, however, activated NF-κB, suggesting tonic inhibition of NF-κB under basal conditions. NO had little or no effects on other nuclear binding proteins such as AP-1 and GATA. Immunoprecipitation studies showed that NO stabilized the NF-κB inhibitor, IκBα, by preventing its degradation from NF-κB. NO also increased the mRNA expression of IκBα, but not NF-κB subunits, p65 or p50, and transfection experiments with a chloramphenicol acetyltransferase reporter gene linked to the IκBα promoter suggested transcriptional induction of IκBα by NO. We propose that the induction and stabilization of IκBα by NO are important mechanisms by which NO inhibits NF-κB and attenuate atherogenesis.
• Peng, H. B., Rajavashisth, T. B., Libby, P., & Liao, J. K. (1995). Nitric oxide inhibits macrophage-colony stimulating factor gene transcription in vascular endothelial cells. Journal of Biological Chemistry, 270(Issue 28). doi:10.1074/jbc.270.28.17050
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  Macrophage-colony stimulating factor (M-CSF) contributes to atherogenesis by regulating macrophage-derived foam cells in atherosclerotic lesions. Here we report that nitric oxide (NO) inhibits the expression of M-CSF in human vascular endothelial cells independent of guanylyl cyclase activation. The induction of M-CSF mRNA expression by either oxidized low density lipoprotein (ox-LDL) or tumor necrosis factor-α (TNFα) was attenuated by NO donors, S- nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and 3- morpholinosydnonimine, but not by cGMP analogues, glutathione, or nitrite. Inhibition of endogenous NO production by N-monomethyl-L-arginine (L-NMA) also increased M-CSF expression in control and TNFα-stimulated cells. Nuclear run-on assays and transfection studies using M-CSF promoter constructs linked to chloramphenicol acetyltransferase reporter gene indicated that NO repressed M-CSF gene transcription through nuclear factor- κB (NF-κB). Electrophoretic mobility shift assays demonstrated that activation of NF-κB by L-NMA, oxLDL, and TNFα was attenuated by GSNO and SNP, but not by glutathione or cGMP analogues. Since the induction of M-CSF expression depends upon NF-κB activation, the ability of NO to inhibit NF- κB activation and M-CSF expression may contribute to some of NO's anti- atherogenic properties.
• Liao, J. K. (1994). Inhibition of Gi proteins by low density lipoprotein attenuates bradykinin-stimulated release of endothelial-derived nitric oxide. Journal of Biological Chemistry, 269(Issue 17). doi:10.1016/s0021-9258(18)99973-4
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  The mechanism(s) by which low density lipoprotein (LDL) attenuates the release of endothelial-derived nitric oxide (EDNO) is not known but may involve inhibition of membrane signal transduction. To test this hypothesis, we treated bovine aortic endothelial cells with LDL (0-500 μg/ml, 72 h) to determine its effect on G proteins (Gi and Gq) which are known to couple the bradykinin receptor to EDNO release. Bradykinin-stimulated EDNO release was measured by perfusion bioassay and showed a maximal 56% decrease which was unaffected by cotreatment with pertussis toxin (PTX, 100 ng/ml, 16 h). Although radioligand binding studies and Western blotting did not detect any significant changes in the amount of bradykinin receptor, αi2, αi3, and αq, both agonist-stimulated GTPase activity and phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis were reduced by LDL treatment (77 and 70%, respectively). When Gi and Gq function in LDL-treated membranes were assessed by PTX and COOH-terminal antisera to αi2 (P4), αi3 (JL14), and αq (Q17), only the Q17 antisera caused a further reduction in GTPase activity and PIP2 hydrolysis while treatment with PTX alone or in combination with the P4 and JL14 antisera had no further inhibitory effect. These results suggest that LDL attenuates EDNO release by preferentially inhibiting the function of Gi.
• Liao, J. K., & Homcy, C. J. (1993). The G proteins of the Gαi and Gαq family couple the bradykinin receptor to the release of endothelium-derived relaxing factor. Journal of Clinical Investigation, 92(Issue 5). doi:10.1172/jci116818
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  Bradykinin stimulates diverse functions in endothelial cells including the release of endothelium-derived relaxing factor (EDRF). Little is known, however, regarding the identity of the G protein(s) involved. Here we demonstrate that G proteins of the Gαi and Gαq family are coupled to the bradykinin receptor (BKR) in bovine aortic endothelial cells by using specific antisera directed against the COOH-terminal region of Gαi2 (P4), Gαi3 (EC), and Gαq (QL). These antisera are specific since their effects are blocked by the decapeptides from which they were derived. The degree of receptor-G protein coupling was assessed by the formation of high affinity agonist binding sites (HABS) and GTP hydrolysis. In a concentration-dependent manner, the QL antisera reduced HABS and GTPase activity by 65 and 60%, respectively, and effectively abolished them in membranes from pertussis toxin-treated cells. The combination of P4 and EC antisera produced a loss of HABS (41%) and GTPase activity (40%) comparable to the effects of pertussis toxin. These findings indicate that Gαi and Gαq proteins mediate the cellular responses to bradykinin in bovine aortic endothelial cells and support the observation that bradykinin-stimulated EDRF release is relatively insensitive to pertussis toxin.
• Liao, J. K., & Homcy, C. J. (1993). The release of endothelium-derived relaxing factor via α2-adrenergic receptor activation is specifically mediated by Giα2. Journal of Biological Chemistry, 268(Issue 26). doi:10.1016/s0021-9258(19)36547-0
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  The purpose of this study is to determine which pertussis toxin-sensitive guanine nucleotide-binding protein (Gi) mediates α2-adrenergic receptor stimulation of endothelium-derived relaxing factor (EDRF) release. Bovine aortic endothelial cells were treated with pertussis toxin (0-100 ng/ml) for 16 h and stimulated with an α2-adrenergic receptor agonist, UK14304, to release EDRF in a bioassay system. Pertussis toxin produced a concentration-dependent decrease in EDRF release with maximal inhibition (80%) occurring at 5 ng/ml. This correlated with a decrease in receptor-G protein coupling as measured by 87% loss of high affinity agonist binding sites and 94% decrease in agonist-stimulated GTPase activity. Immunoprecipitation of [32P]NAD-ribosylated membranes using specific Gi protein antisera demonstrated that complete ADP-ribosylation of Giα2 occurred at 5 ng/ml compared to 30 ng/ml for Giα3. When bovine aortic endothelial cell membranes were treated with carboxyl terminus-directed antisera to Giα2 (P4) and Giα3 (EC), the P4 antisera abolished 86% of the high affinity agonist binding sites and 93% decrease in agonist-stimulated GTPase activity, while the EC antisera had minimal effect (12%). These results indicate that Giα2 mediates most of the EDRF released via the α2-adrenergic receptor.
• Liao, J. K., Bettmann, M. A., Sandor, T., Tucker, J. I., Coleman, S. M., & Creager, M. A. (1991). Differential impairment of vasodilator responsiveness of peripheral resistance and conduit vessels in humans with atherosclerosis. Circulation Research, 68(Issue 4). doi:10.1161/01.res.68.4.1027
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  The purpose of this study was to assess the effect of atherosclerosis on the regulation of limb blood flow. To examine this issue, the reactivity of resistance and conduit vessels was evaluated in 11 patients with peripheral atherosclerotic disease and six control subjects. Responsiveness of resistance vessels was measured by venous occlusion plethysmography. Responsiveness of conduit vessels was determined by quantitative angiography to measure the diameter of the superficial femoral artery. To distinguish endothelium-dependent vasodilation from that caused by direct smooth muscle relaxation, each participant received intra-arterial infusions of methacholine and nitroprusside, respectively. Flow-mediated dilation of the superficial femoral artery was determined during reactive hyperemia. Vasoconstrictor function was determined by the infusion of phenylephrine. Methacholine reduced calf vascular resistance in the control subjects but not in the patients with atherosclerosis (-64 ± 11% versus 6 ± 18%, p < 0.01). Nitroprusside decreased calf vascular resistance comparably in each group (-51 ± 5% versus -42 ± 4%, p = NS). The vasoconstrictor effect of phenylephrine was similar in each group (105 ± 30% versus 108 ± 22%, p = NS). In the superficial femoral artery, the vasodilator responses to both methacholine (20 ± 4% versus 1 ± 4%, p < 0.05) and nitroprusside (19 ± 4% versus 5 ± 4%, p < 0.05) were blunted in the atherosclerotic patients as was the vasoconstrictive response to phenylephrine (-15 ± 1% versus -1 ± 5%, p < 0.05). Reactive hyperemia induced a fivefold increase in calf blood flow in both control subjects (1.6 ± 0.2 to 11.1 ± 2.9 ml/100 ml/min, p < 0.05) and atherosclerotic patients (2.2 ± 0.3 to 10.0 ± 1.5 ml/100 ml/min, p < 0.05). During this flow augmentation, the superficial femoral artery dilated in control subjects (7.5 ± 0.8 to 8.2 ± 0.3 mm, p < 0.05) but not in atherosclerotic patients (6.0 ± 0.5 to 6.1 ± 0.5 mm, p = NS). Therefore, in humans with peripheral atherosclerosis, endothelium-dependent vasodilation in resistance vessels is impaired. Atherosclerotic conduit vessels are essentially unresponsive to vasodilator and vasoconstrictor stimuli, implicating functional abnormalities intrinsic to the vascular smooth muscle. These disturbances in vasomotor control may adversely affect blood flow regulation in humans with peripheral atherosclerosis and contribute to symptoms of claudication that occur in this disorder.
• Liao, J. K., Cockrill, B. A., & Yurchak, P. M. (1991). Acute myocardial infarction after ergonovine administration for uterine bleeding. The American Journal of Cardiology, 68(Issue 8). doi:10.1016/0002-9149(91)90669-c
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  Acute myocardial infarction during pregnancy and in the postpartum period is a rare occurrence of uncertain etiology.1 It has been reported in women with atherosclerotic coronary artery disease, diabetes mellitus and systemic hypertension.2 In women with normal coronary angiograms, however, no clear and consistent etiology can be found. A few cases did involve patients receiving ergot derivatives, but the events were poorly documented and established only by retrospective association. We report a patient who sustained an acute myocardial infarction after receiving methylergonovine for excessive uterine bleeding after a first trimester spontaneous abortion. The cardiac event was promptly recognized and treated, resulting in a relatively benign postmyocardial infarction course. © 1991.
• Palusinski, O., Liao, J., Prince, J., & Cangellaris, A. (1990). Simulation of transients in VLSI packaging interconnections. IEEE Transactions on Components, Hybrids, and Manufacturing Technology, 13(1), 160-166. doi:10.1109/33.52865

Proceedings Publications

• Liao, J. K. (2003). Cross-coupling between the oestrogen receptor and phosphoinositide 3-kinase. In N/A, 31.
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  Gender differences in cardiovascular mortality are well documented and oestrogen replacement therapy in post-menopausal women is associated with improved outcomes from cardiovascular events. Indeed, oestrogen therapy has been shown to restore endothelial function in post-menopausal women and reduce the development of atherosclerotic lesions. Despite extensive evidence on the beneficial effect of oestrogen, relatively little is known regarding the molecular signalling mechanism(s) by which oestrogen exerts some of its vascular effects. While the nuclear function of the oestrogen receptor is clearly established, previous studies regarding the membrane and cytoplasmic actions of oestrogen remain inconclusive. Cross-coupling of the oestrogen receptor to phosphoinositide 3-kinase signalling suggests a potentially critical non-nuclear action of the oestrogen receptor and considerably broadens our understanding of the cellular effects of oestrogen.
• De Caterina, R., Liao, J. K., & Libby, P. (2000). Fatty acid modulation of endothelial activation. In N/A, 71.
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  Dietary balance of long-chain fatty acids may influence processes involving leukocyte-endothelial interactions, such as atherogenesis and inflammation, that involve increased endothelial expression of leukocyte adhesion molecules, or endothelial activation. We compared the ability of various saturated, monounsaturated, and polyunsaturated fatty acids to modulate endothelial activation. Consumption of the n-3 fatty acid docosahexaenoic acid (DHA; 22:6n-3) reduced endothelial expression of vascular cell adhesion molecule 1 (VCAM-1), E-selectin, intercellular adhesion molecule 1 (ICAM-1), interleukin 6 (IL-6), and IL-8 in response to IL-1, IL-4, tumor necrosis factor, or bacterial endotoxin, with a half- maximal inhibitory concentration (IC50) of 1-25 μmol, ie, in the range of nutritionally achievable plasma concentrations. The magnitude of this effect paralleled its incorporation into cellular phospholipids DHA also reduced the adhesion of human monocytes and monocytic U937 cells to cytokine-stimulated endothelial cells. These effects were accompanied by a reduction in VCAM-1 messenger RNA, indicating a pretranslational effect. To assess structural fatty acid determinants of VCAM-1 inhibitory activity, we compared various saturated, monounsaturated, and n-6 and n-3 polyunsaturated fatty acids for their VCAM-1 inhibitory activity. Saturated fatty acids did not inhibit cytokine-induced expression of adhesion molecules. However, a progressive increase in inhibitory activity was observed with dietary intake of fatty acids with the same chain length but increasing double bonds, ie, from monounsaturated to n-6 and, further, to n-3 fatty acids. Thus, the greater number of double bonds seems critical for the greater activity of n-3 compared with n-6 fatty acids in inhibiting endothelial activation. These properties are likely to be relevant to the antiatherogenic and antiinflammatory properties of n-3 fatty acids.
• Liao, J. K. (1998). Endothelium and acute coronary syndromes. In N/A, 44.
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  The vascular endothelium is a dynamic endocrine organ that regulates contractile, secretory, and mitogenic activities in the vessel wall and hemostatic processes within the vascular lumen. Risk factors for atherosclerosis such as cigarette smoking, hypertension, and increased serum lipid concentrations impair endothelial function and lead to the development of atherosclerotic vessels, which cause acute coronary syndromes. Atherosclerotic vessels progress from scattered foam cells to complex lesions with a lipid core and fibrous cap. Factors that weaken and cause the rupture of the fibrous cap will expose circulating blood products to the procoagulant materials in the lipid core. Thrombosis and subsequent remodeling of the unstable plaque may be catastrophic or clinically silent depending on the degree of vascular occlusion and availability of collateral blood flow. Evidence is presented that supports the view that endothelial dysfunction is an early marker of atherosclerosis and an important contributor to the atherogenic process.
• Libby, P., Sukhova, G., Lee, R. T., & Liao, J. K. (1997). Molecular biology of atherosclerosis. In N/A, 62.
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  For much of the last century, the development of arteriosclerosis was regarded as an inevitable degenerative process. Osler stated: 'the stability of tubing of any sort depends on the sort of material used; and so it is with the human being. With the poor variety of elastic and muscular fibers in the blood vessels, some are unable to resist the wear and tear of daily life' [1]. Recently, thinking regarding atherogenesis has evolved from vague concepts of inevitable degeneration to a more precise sequence of molecular and cellular events. As we enhance our understanding of its fundamental mechanisms, we can begin to approach atherogenesis as a modifiable process. Eventually, mastery of the cell and molecular biologies of atherosclerosis may permit the development of novel strategies for mitigating this prevalent disease. Atherogenesis in humans generally occurs over many years, often measured in decades. Lesion initiation may occur as early as childhood. Lesion evolution and growth varies according to heredity, gender, and well- defined risk factors. Complications of atheroma that usually underlie the acute manifestations of this disease may come about suddenly. Some individuals with atherosclerosis may never have symptoms, others may have only chronic stable manifestations, and yet others may experience fatal or life-threatening acute events without having passed through a phase of chronic symptoms. This review will consider in turn each of the three major phases in the life history of an atheroma. We will discuss aspects of lesion initiation, progression, and complication. Rather than attempting a comprehensive overview, we will focus primarily on selected examples where new information sheds light on potential molecular mechanisms underlying these pathologic processes.