Won Hee Lee

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• Ong, S., Lee, W., Zhou, Y., & Wu, J. (2018). Mining exosomal MicroRNAs from human-induced pluripotent stem cells-derived cardiomyocytes for cardiac regeneration. In Methods in Molecular Biology. doi:10.1007/978-1-4939-7601-0_10
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  Myocardial infarction is the leading cause of morbidity and mortality worldwide. Recent advances in cardiac regenerative therapy have allowed for novel modalities in replenishing the damaged myocardium. However, poor long-term engraftment and survival of transplanted cells have largely precluded effective cell replacement. As an alternative to direct cell replacement, the release of paracrine protective factors may be a more plausible effector for cardioprotection which may partially be mediated through secretion of microvesicles, or exosomes, that contribute to cell-cell communication. In this chapter, we describe the isolation of exosomes from induced pluripotent stem cells-derived cardiomyocytes for subsequent microRNA profiling for a better understanding of the biological cargo contained within exosomes.

Journals/Publications

• Yan, G., Han, Z., Kwon, Y., Jousma, J., Nukala, S. B., Prosser, B. L., Du, X., Pinho, S., Ong, S. B., Lee, W. H., & Ong, S. G. (2024). Integrated Stress Response Potentiates Ponatinib-Induced Cardiotoxicity. Circulation research, 134(5), 482-501.
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  Mitochondrial dysfunction is a primary driver of cardiac contractile failure; yet, the cross talk between mitochondrial energetics and signaling regulation remains obscure. Ponatinib, a tyrosine kinase inhibitor used to treat chronic myeloid leukemia, is among the most cardiotoxic tyrosine kinase inhibitors and causes mitochondrial dysfunction. Whether ponatinib-induced mitochondrial dysfunction triggers the integrated stress response (ISR) to induce ponatinib-induced cardiotoxicity remains to be determined.
• Jousma, J., Park, J., Han, Z., Yan, G., Nukala, S. B., Kwon, Y., Lee, W. H., Jiang, Y., & Ong, S. (2023).

  Abstract P2084: Adipose Tissues Thermogenesis Provides Cardioprotection Against Diastolic Dysfunction Developed In An Obesity-Induced HFpEF Model

  . Circulation Research, 133(Suppl_1). doi:10.1161/res.133.suppl_1.p2084
• Liu, C. W., Le, H. H., Denaro Iii, P., Dai, Z., Shao, N. Y., Ong, S. G., & Lee, W. H. (2023). E-cigarettes induce dysregulation of autophagy leading to endothelial dysfunction in pulmonary arterial hypertension. Stem cells (Dayton, Ohio).
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  Given the increasing popularity of electronic cigarettes (e-cigs), it is imperative to evaluate the potential health risks of e-cigs, especially in users with preexisting health concerns such as pulmonary arterial hypertension (PAH). The aim of the present study was to investigate whether differential susceptibility exists between healthy and PAH patients to e-cig exposure and the molecular mechanisms contributing to it. Patient-specific induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from healthy individuals and PAH patients were used to investigate whether e-cig contributes to the pathophysiology of PAH and affects EC homeostasis in PAH. Our results showed that PAH iPSC-ECs showed a greater amount of damage than healthy iPSC-ECs upon e-cig exposure. Transcriptomic analyses revealed that differential expression of Akt3 may be responsible for increased autophagic flux impairment in PAH iPSC-ECs, which underlies increased susceptibility upon e-cig exposure. Moreover, knockdown of Akt3 in healthy iPSC-ECs significantly induced autophagic flux impairment and endothelial dysfunction, which further increased with e-cig treatment, thus mimicking the PAH cell phenotype after e-cig exposure. In addition, functional disruption of mTORC2 by knocking down Rictor in PAH iPSC-ECs caused autophagic flux impairment, which was mediated by downregulation of Akt3. Finally, pharmacological induction of autophagy via direct inhibition of mTORC1 and indirect activation of mTORC2 with rapamycin reverses e-cig-induced decreased Akt3 expression, endothelial dysfunction, autophagic flux impairment, and decreased cell viability and migration in PAH iPSC-ECs. Taken together, these data suggest a potential link between autophagy and Akt3-mediated increased susceptibility to e-cig in PAH.
• Nukala, S. B., Jousma, J., Yan, G., Han, Z., Kwon, Y., Cho, Y., Liu, C., Gagnon, K., Pinho, S., Rehman, J., Shao, N. Y., Ong, S. B., Lee, W. H., & Ong, S. G. (2023). Modulation of lncRNA links endothelial glycocalyx to vascular dysfunction of tyrosine kinase inhibitor. Cardiovascular research.
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  Novel cancer therapies leading to increased survivorship of cancer patients have been negated by a concomitant rise in cancer therapies-related cardiovascular toxicities. Sunitinib, a first line multi receptor tyrosine kinase inhibitor (TKI), has been reported to cause vascular dysfunction although the initiating mechanisms contributing to this side effect remain unknown. Long non-coding RNAs (lncRNAs) are emerging regulators of biological processes in endothelial cells (ECs); however, their roles in cancer therapies-related vascular toxicities remain underexplored.
• Yi, D., Liu, B., Ding, H., Li, S., Li, R., Pan, J., Ramirez, K., Xia, X., Kala, M., Ye, Q., Lee, W. H., Frye, R. E., Wang, T., Zhao, Y., Knox, K. S., Glembotski, C. C., Fallon, M. B., & Dai, Z. (2023).

  E2F1 Mediates SOX17 Deficiency–Induced Pulmonary Hypertension

  . Hypertension, 80(11), 2357-2371. doi:10.1161/hypertensionaha.123.21241
• Jousma, J., Han, Z., Yan, G., Nukala, S. B., Kwon, Y., Thi Le, H. H., Li, Y., Ong, S. B., Lee, W. H., & Ong, S. G. (2022). Alteration of the N6-methyladenosine epitranscriptomic profile in synthetic phthalate-treated human induced pluripotent stem cell-derived endothelial cells. Epigenomics, 14(19), 1139-1155.
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  This study aimed to characterize the N-methyladenosine epitranscriptomic profile induced by mono(2-ethylhexyl) phthalate (MEHP) exposure using a human-induced pluripotent stem cell-derived endothelial cell model. A multiomic approach was employed by performing RNA sequencing in parallel with an N-methyladenosine-specific microarray to identify mRNAs, lncRNAs, and miRNAs affected by MEHP exposure. An integrative multiomic analysis identified relevant biological features affected by MEHP, while functional assays provided a phenotypic characterization of these effects. Transcripts regulated by the epitranscriptome were validated with quantitative PCR and methylated RNA immunoprecipitation. The authors' profiling of the epitranscriptome expands the scope of toxicological insights into known environmental toxins to under surveyed cellular contexts and emerging domains of regulation and is, therefore, a valuable resource to human health.
• Nukala, S. B., Jousma, J., Cho, Y., Lee, W. H., & Ong, S. G. (2022). Long non-coding RNAs and microRNAs as crucial regulators in cardio-oncology. Cell & bioscience, 12(1), 24.
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  Cancer is one of the leading causes of morbidity and mortality worldwide. Significant improvements in the modern era of anticancer therapeutic strategies have increased the survival rate of cancer patients. Unfortunately, cancer survivors have an increased risk of cardiovascular diseases, which is believed to result from anticancer therapies. The emergence of cardiovascular diseases among cancer survivors has served as the basis for establishing a novel field termed cardio-oncology. Cardio-oncology primarily focuses on investigating the underlying molecular mechanisms by which anticancer treatments lead to cardiovascular dysfunction and the development of novel cardioprotective strategies to counteract cardiotoxic effects of cancer therapies. Advances in genome biology have revealed that most of the genome is transcribed into non-coding RNAs (ncRNAs), which are recognized as being instrumental in cancer, cardiovascular health, and disease. Emerging studies have demonstrated that alterations of these ncRNAs have pathophysiological roles in multiple diseases in humans. As it relates to cardio-oncology, though, there is limited knowledge of the role of ncRNAs. In the present review, we summarize the up-to-date knowledge regarding the roles of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in cancer therapy-induced cardiotoxicities. Moreover, we also discuss prospective therapeutic strategies and the translational relevance of these ncRNAs.
• Joshi, S., Lee, W. H., Chen, P., Serpooshan, V., & Yang, H. (2021). Editorial: 3D Cell Culture Systems for Cardiovascular Tissue Engineering: Modelling and Regenerative Therapies. Frontiers in cardiovascular medicine, 8, 675676.
• Klionsky, D. J., Abdel-Aziz, A. K., Abdelfatah, S., Abdellatif, M., Abdoli, A., Abel, S., Abeliovich, H., Abildgaard, M. H., Abudu, Y. P., Acevedo-Arozena, A., Adamopoulos, I. E., Adeli, K., Adolph, T. E., Adornetto, A., Aflaki, E., Agam, G., Agarwal, A., Aggarwal, B. B., Agnello, M., , Agostinis, P., et al. (2021). Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy, 17(1), 1-382.
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  In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
• Le, H. H., Liu, C. W., Denaro, P., Jousma, J., Shao, N. Y., Rahman, I., & Lee, W. H. (2021). Genome-wide differential expression profiling of lncRNAs and mRNAs in human induced pluripotent stem cell-derived endothelial cells exposed to e-cigarette extract. Stem cell research & therapy, 12(1), 593.
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  Electronic-cigarette (e-cig) usage, particularly in the youth population, is a growing concern. It is known that e-cig causes endothelial dysfunction, which is a risk factor for the development of cardiovascular diseases; however, the mechanisms involved remain unclear. We hypothesized that long noncoding RNAs (lncRNAs) may play a role in e-cig-induced endothelial dysfunction.
• Lee, W. H., Nukala, S. B., Kwon, Y., Jousma, J., Yan, G., Han, Z., Cho, Y., Shao, N., & Ong, S. (2021). Abstract 14018: lncRNA HAS2-AS1 Regulates Sunitinib-Induced Endothelial Dysfunction by Preserving the Endothelial Glycocalyx. Circulation, 144(Suppl_1). doi:10.1161/circ.144.suppl_1.14018
• Jang, H. R., Cho, H. J., Zhou, Y., Shao, N. Y., Lee, K., Le, H. H., Jeon, J., Lee, J. E., Huh, W., Ong, S. G., Lee, W. H., & Kim, Y. G. (2020). Modeling Uremic Vasculopathy With Induced Pluripotent Stem Cell-Derived Endothelial Cells as a Drug Screening System. Frontiers in cell and developmental biology, 8, 618796.
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  Cardiovascular complications are the leading cause of mortality in patients with chronic kidney disease (CKD). Uremic vasculopathy plays a crucial role in facilitating the progression of cardiovascular complications in advanced CKD. However, the improvement of conventional research methods could provide further insights into CKD. In this study, we aimed to develop a novel model of uremic vasculopathy as a potential drug screening system. The effects of uremic serum and different combinations of uremic toxins on induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) of a normal control and a CKD patient were investigated using several functional assays. We found that a mixture of uremic toxins composed of high urea, creatinine, uric acid, and indoxyl sulfate exerted deleterious effects on normal control iPSC-ECs that were comparable to uremic serum by increasing reactive oxygen species and apoptosis, as well as suppression of tube formation. Additional characterization revealed a potential involvement of dysregulated TGF-β signaling as treatment with either losartan or TGF-β inhibitors led to the attenuation of adverse effects induced by uremic toxins. Importantly, impaired wound healing potential seen in CKD patient-specific iPSC-ECs was rescued by treatment with losartan and TGF-β inhibitors. Our study demonstrated that simplified uremic toxin mixtures can simulate the uremic micromilieu reproducibly and CKD patient-specific iPSC-ECs can potentially recapitulate susceptibility to uremic vasculopathy. This novel model of uremic vasculopathy may provide a new research tool as a drug screening system.
• Kwon, Y., Nukala, S. B., Srivastava, S., Miyamoto, H., Ismail, N. I., Jousma, J., Rehman, J., Ong, S. B., Lee, W. H., & Ong, S. G. (2020). Detection of viral RNA fragments in human iPSC cardiomyocytes following treatment with extracellular vesicles from SARS-CoV-2 coding sequence overexpressing lung epithelial cells. Stem cell research & therapy, 11(1), 514.
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  Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global pandemic. The prevalence/severity of COVID-19 is higher among patients with cardiovascular risk factors. Despite the expression of angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV-2 infection, in cardiomyocytes, there has been no conclusive evidence of direct viral infection although the presence of viral genome within COVID-19 patients' hearts has been reported. Here, we overexpressed SARS-CoV-2 genes in A549 lung epithelial cells. We then isolated extracellular vesicles (EVs) and detected the presence of viral RNA within these EVs. We observed that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are receptive to these EVs, and viral genes were detectable in the cardiomyocytes. Accordingly, the uptake of viral RNA-harboring EVs led to an upregulation of inflammation-related genes in hiPSC-CMs. Thus, our findings indicate that SARS-CoV-2 RNA containing EVs represents an indirect route of viral RNA entry into cardiomyocytes.
• Guo, H., Tian, L., Zhang, J. Z., Kitani, T., Paik, D. T., Lee, W. H., & Wu, J. C. (2019). Single-Cell RNA Sequencing of Human Embryonic Stem Cell Differentiation Delineates Adverse Effects of Nicotine on Embryonic Development. Stem cell reports, 12(4), 772-786.
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  Nicotine, the main chemical constituent of tobacco, is highly detrimental to the developing fetus by increasing the risk of gestational complications and organ disorders. The effects of nicotine on human embryonic development and related mechanisms, however, remain poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) of human embryonic stem cell (hESC)-derived embryoid body (EB) in the presence or absence of nicotine. Nicotine-induced lineage-specific responses and dysregulated cell-to-cell communication in EBs, shedding light on the adverse effects of nicotine on human embryonic development. In addition, nicotine reduced cell viability, increased reactive oxygen species (ROS), and altered cell cycling in EBs. Abnormal Ca signaling was found in muscle cells upon nicotine exposure, as verified in hESC-derived cardiomyocytes. Consequently, our scRNA-seq data suggest direct adverse effects of nicotine on hESC differentiation at the single-cell level and offer a new method for evaluating drug and environmental toxicity on human embryonic development in utero.
• Lee, W. H., Ong, S. G., Zhou, Y., Tian, L., Bae, H. R., Baker, N., Whitlatch, A., Mohammadi, L., Guo, H., Nadeau, K. C., Springer, M. L., Schick, S. F., Bhatnagar, A., & Wu, J. C. (2019). Modeling Cardiovascular Risks of E-Cigarettes With Human-Induced Pluripotent Stem Cell-Derived Endothelial Cells. Journal of the American College of Cardiology, 73(21), 2722-2737.
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  Electronic cigarettes (e-cigarettes) have experienced a tremendous increase in use. Unlike cigarette smoking, the effects of e-cigarettes and their constituents on mediating vascular health remain understudied. However, given their increasing popularity, it is imperative to evaluate the health risks of e-cigarettes, including the effects of their ingredients, especially nicotine and flavorings.
• Ong, S. B., Lee, W. H., Shao, N. Y., Ismail, N. I., Katwadi, K., Lim, M. M., Kwek, X. Y., Michel, N. A., Li, J., Newson, J., Tahmasebi, S., Rehman, J., Kodo, K., Jang, H. R., & Ong, S. G. (2019). Calpain Inhibition Restores Autophagy and Prevents Mitochondrial Fragmentation in a Human iPSC Model of Diabetic Endotheliopathy. Stem cell reports, 12(3), 597-610.
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  The relationship between diabetes and endothelial dysfunction remains unclear, particularly the association with pathological activation of calpain, an intracellular cysteine protease. Here, we used human induced pluripotent stem cells-derived endothelial cells (iPSC-ECs) to investigate the effects of diabetes on vascular health. Our results indicate that iPSC-ECs exposed to hyperglycemia had impaired autophagy, increased mitochondria fragmentation, and was associated with increased calpain activity. In addition, hyperglycemic iPSC-ECs had increased susceptibility to cell death when subjected to a secondary insult-simulated ischemia-reperfusion injury (sIRI). Importantly, calpain inhibition restored autophagy and reduced mitochondrial fragmentation, concurrent with maintenance of ATP production, normalized reactive oxygen species levels and reduced susceptibility to sIRI. Using a human iPSC model of diabetic endotheliopathy, we demonstrated that restoration of autophagy and prevention of mitochondrial fragmentation via calpain inhibition improves vascular integrity. Our human iPSC-EC model thus represents a valuable platform to explore biological mechanisms and new treatments for diabetes-induced endothelial dysfunction.
• Cho, H., Lee, W., Hwang, O., Sonntag, W., & Lee, Y. (2017). Role of NADPH oxidase in radiation-induced pro-oxidative and pro-inflammatory pathways in mouse brain. International Journal of Radiation Biology, 93(11). doi:10.1080/09553002.2017.1377360
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  Purpose: The present study was designed to investigate our hypothesis that NADPH oxidase plays a role in radiation-induced pro-oxidative and pro-inflammatory environments in the brain. Materials and methods: C57BL/6 mice received either fractionated whole brain irradiation or sham-irradiation. The mRNA expression levels of pro-inflammatory mediators, such as TNF-α and MCP-1, were determined by quantitative real-time RT-PCR. The protein expression levels of TNF-α, MCP-1, NOX-2 and Iba1 were detected by immunofluorescence staining. The levels of ROS were visualized by in situ DHE fluorescence staining. Results: A significant up-regulation of mRNA and protein expression levels of TNF-α and MCP-1 was observed in irradiated mouse brains. Additionally, immunofluorescence staining of Iba1 showed a marked increase of microglial activation in mouse brain after irradiation. Moreover, in situ DHE fluorescence staining revealed that fractionated whole brain irradiation significantly increased production of ROS. Furthermore, a significant increase in immunoreactivity of NOX-2 was detected in mouse brain after irradiation. On the contrary, an enhanced ROS generation in mouse brain after irradiation was markedly attenuated in the presence of NOX inhibitors or NOX-2 neutralizing antibody. Conclusions: These results suggest that NOX-2 may play a role in fractionated whole brain irradiation-induced pro-oxidative and pro-inflammatory pathways in mouse brain.
• Lee, W., Chen, W., Shao, N., Xiao, D., Qin, X., Baker, N., Bae, H., Wei, T., Wang, Y., Shukla, P., Wu, H., Kodo, K., Ong, S., & Wu, J. (2017). Comparison of Non-Coding RNAs in Exosomes and Functional Efficacy of Human Embryonic Stem Cell- versus Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Stem Cells, 35(10). doi:10.1002/stem.2669
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  Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived CMs (iPSC-CMs) can serve as unlimited cell sources for cardiac regenerative therapy. However, the functional equivalency between human ESC-CMs and iPSC-CMs for cardiac regenerative therapy has not been demonstrated. Here, we performed a head-to-head comparison of ESC-CMs and iPSC-CMs in their ability to restore cardiac function in a rat myocardial infarction (MI) model as well as their exosomal secretome. Human ESCs and iPSCs were differentiated into CMs using small molecule inhibitors. Fluorescence-activated cell sorting analysis confirmed ∼85% and ∼83% of CMs differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T. At a single-cell level, both cell types displayed similar calcium handling and electrophysiological properties, with gene expression comparable with the human fetal heart marked by striated sarcomeres. Sub-acute transplantation of ESC-CMs and iPSC-CMs into nude rats post-MI improved cardiac function, which was associated with increased expression of angiogenic genes in vitro following hypoxia. Profiling of exosomal microRNAs (miRs) and long non-coding RNAs (lncRNAs) revealed that both groups contain an identical repertoire of miRs and lncRNAs, including some that are known to be cardioprotective. We demonstrate that both ESC-CMs and iPSC-CMs can facilitate comparable cardiac repair. This is advantageous because, unlike allogeneic ESC-CMs used in therapy, autologous iPSC-CMs could potentially avoid immune rejection when used for cardiac cell transplantation in the future. Stem Cells 2017;35:2138–2149.
• Hnatiuk, A., Ong, S., Olea, F., Locatelli, P., Riegler, J., Lee, W., Jen, C., Lorenzi, A., Laguens, R., Wu, J., Crottogini, A., & Giménez, C. (2016). Allogeneic mesenchymal stromal cells overexpressing mutant human Hypoxia-inducible factor 1-α (HIF1-α) in an ovine model of acute myocardial infarction. ournal of the American Heart Association, 5(7). doi:10.1161/JAHA.116.003714
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  Background-Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia-inducible factor 1-α (HIF1-α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen-resistant HIF1-α would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI. Methods and Results-Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1-α (BMMSC-HIF) were injected in the peri-infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P < 0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2-fold (P < 0.001) in the presence of markedly decreased end-systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo-treated animals (n=6), neither parameters changed over time. HIF1-α-transfected BMMSCs (BMMSC-HIF) induced angio-/arteriogenesis and decreased apoptosis by HIF1-mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin-1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long-term retention of BMMSC-HIF. Conclusions-Intramyocardial delivery of BMMSC-HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1-mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.
• Hu, S., Zhao, M., Jahanbani, F., Shao, N., Lee, W., Chen, H., Snyder, M., & Wu, J. (2016). Effects of cellular origin on differentiation of human induced pluripotent stem cell-derived endothelial cells. Journal of Clinical Investigation Insight, 1(8). doi:10.1172/jci.insight.85558
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  Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC-derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC-ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC-ECs maintained a higher CD31+ population than FB-iPSC-ECs and CPC-iPSC-ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation.
• Lee, W., Nguyen, P., Fleischmann, D., & Wu, J. (2016). DNA damage-Associated biomarkers in studying individual sensitivity to low-dose radiation from cardiovascular imaging. European Heart Journal, 37(40). doi:10.1093/eurheartj/ehw206
• Riegler, J., Ebert, A., Qin, X., Shen, Q., Wang, M., Ameen, M., Kodo, K., Ong, S., Lee, W., Lee, G., Neofytou, E., Gold, J., Connolly, A., & Wu, J. (2016). Comparison of Magnetic Resonance Imaging and Serum Biomarkers for Detection of Human Pluripotent Stem Cell-Derived Teratomas. Stem Cell Reports, 6(2). doi:10.1016/j.stemcr.2015.12.008
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  The use of cells derived from pluripotent stem cells (PSCs) for regenerative therapies confers a considerable risk for neoplastic growth and teratoma formation. Preclinical and clinical assessment of such therapies will require suitable monitoring strategies to understand and mitigate these risks. Here we generated human-induced pluripotent stem cells (iPSCs), selected clones that continued to express reprogramming factors after differentiation into cardiomyocytes, and transplanted these cardiomyocytes into immunocompromised rat hearts post-myocardial infarction. We compared magnetic resonance imaging (MRI), cardiac ultrasound, and serum biomarkers for their ability to delineate teratoma formation and growth. MRI enabled the detection of teratomas with a volume >8 mm3. A combination of three plasma biomarkers (CEA, AFP, and HCG) was able to detect teratomas with a volume >17 mm3 and with a sensitivity of more than 87%. Based on our findings, a combination of serum biomarkers with MRI screening may offer the highest sensitivity for teratoma detection and tracking.
• Lee, W., Nguyen, P., Hu, S., Liang, G., Ong, S., Han, L., Sanchez-Freire, V., Lee, A., Vasanawala, M., Segall, G., & Wu, J. (2015). Variable activation of the DNA damage response pathways in patients undergoing single-photon emission computed tomography myocardial perfusion imaging. Circulation Cardiovascular Imaging, 8(2). doi:10.1161/CIRCIMAGING.114.002851
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  Background: Although single-photon emission computed tomography myocardial perfusion imaging (SPECT MPI) has improved the diagnosis and risk stratification of patients with suspected coronary artery disease, it remains a primary source of low-dose radiation exposure for cardiac patients. To determine the biological effects of low-dose radiation fromSPECT MPI, we measured the activation of the DNA damage response pathways using quantitative flow cytometry and single-cell gene expression profiling. Methods and Results: Blood samples were collected from patients before and after SPECT MPI (n=63). Overall, analysis of all recruited patients showed no marked differences in the phosphorylation of proteins (H2AX, protein 53, and ataxia telangiectasia mutated) after SPECT. The majority of patients also had either downregulated or unchanged expression in DNA damage response genes at both 24 and 48 hours post-SPECT. Interestingly, a small subset of patients with increased phosphorylation had significant upregulation of genes associated with DNA damage, whereas those with no changes in phosphorylation had significant downregulation or no difference, suggesting that some patients may potentially be more sensitive to low-dose radiation exposure. Conclusions: Our findings showed that SPECT MPI resulted in a variable activation of the DNA damage response pathways. Although only a small subset of patients had increased protein phosphorylation and elevated gene expression postimaging, continued care should be taken to reduce radiation exposure to both the patients and operators.
• Nguyen, P., Lee, W., Li, Y., Hong, W., Hu, S., Chan, C., Liang, G., Nguyen, I., Ong, S., Churko, J., Wang, J., Altman, R., Fleischmann, D., & Wu, J. (2015). Assessment of the radiation effects of cardiac CT angiography using protein and genetic biomarkers. Journal of the American College of Cardiology Cardiovascular Imaging, 8(8). doi:10.1016/j.jcmg.2015.04.016
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  Objectives The purpose of this study was to evaluate whether radiation exposure from cardiac computed tomographic angiography (CTA) is associated with deoxyribonucleic acid (DNA) damage and whether damage leads to programmed cell death and activation of genes involved in apoptosis and DNA repair. Background Exposure to radiation from medical imaging has become a public health concern, but whether it causes significant cell damage remains unclear. Methods We conducted a prospective cohort study in 67 patients undergoing cardiac CTA between January 2012 and December 2013 in 2 U.S. medical centers. Median blood radiation exposure was estimated using phantom dosimetry. Biomarkers of DNA damage and apoptosis were measured by flow cytometry, whole genome sequencing, and single cell polymerase chain reaction. Results The median dose length product was 1,535.3 mGy·cm (969.7 to 2,674.0 mGy·cm). The median radiation dose to the blood was 29.8 mSv (18.8 to 48.8 mSv). Median DNA damage increased 3.39% (1.29% to 8.04%, p < 0.0001) and median apoptosis increased 3.1-fold (interquartile range [IQR]: 1.4- to 5.1-fold, p < 0.0001) post-radiation. Whole genome sequencing revealed changes in the expression of 39 transcription factors involved in the regulation of apoptosis, cell cycle, and DNA repair. Genes involved in mediating apoptosis and DNA repair were significantly changed post-radiation, including DDB2 (1.9-fold [IQR: 1.5- to 3.0-fold], p < 0.001), XRCC4 (3.0-fold [IQR: 1.1- to 5.4-fold], p = 0.005), and BAX (1.6-fold [IQR: 0.9- to 2.6-fold], p < 0.001). Exposure to radiation was associated with DNA damage (odds ratio [OR]: 1.8 [1.2 to 2.6], p = 0.003). DNA damage was associated with apoptosis (OR: 1.9 [1.2 to 5.1], p < 0.0001) and gene activation (OR: 2.8 [1.2 to 6.2], p = 0.002). Conclusions Patients exposed to >7.5 mSv of radiation from cardiac CTA had evidence of DNA damage, which was associated with programmed cell death and activation of genes involved in apoptosis and DNA repair.
• Ong, S., Huber, B., Lee, W., Kodo, K., Ebert, A., Ma, Y., Nguyen, P., Diecke, S., Chen, W., & Wu, J. (2015). Microfluidic single-cell analysis of transplanted human induced pluripotent stem cell-derived cardiomyocytes after acute myocardial infarction. Circulation, 132(8). doi:10.1161/CIRCULATIONAHA.114.015231
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  Background - Human induced pluripotent stem cells (iPSCs) are attractive candidates for therapeutic use, with the potential to replace deficient cells and to improve functional recovery in injury or disease settings. Here, we test the hypothesis that human iPSC-derived cardiomyocytes (iPSC-CMs) can secrete cytokines as a molecular basis to attenuate adverse cardiac remodeling after myocardial infarction. Methods and Results - Human iPSCs were generated from skin fibroblasts and differentiated in vitro with a small molecule-based protocol. Troponin+ iPSC-CMs were confirmed by immunohistochemistry, quantitative polymerase chain reaction, fluorescence-activated cell sorting, and electrophysiological measurements. Afterward, 2×106 iPSC-CMs derived from a cell line transduced with a vector expressing firefly luciferase and green fluorescent protein were transplanted into adult NOD/SCID mice with acute left anterior descending artery ligation. Control animals received PBS injection. Bioluminescence imaging showed limited engraftment on transplantation into ischemic myocardium. However, magnetic resonance imaging of animals transplanted with iPSC-CMs showed significant functional improvement and attenuated cardiac remodeling compared with PBS-treated control animals. To understand the underlying molecular mechanism, microfluidic single-cell profiling of harvested iPSC-CMs, laser capture microdissection of host myocardium, and in vitro ischemia stimulation were used to demonstrate that the iPSC-CMs could release significant levels of proangiogenic and antiapoptotic factors in the ischemic microenvironment. Conclusions - Transplantation of human iPSC-CMs into an acute mouse myocardial infarction model can improve left ventricular function and attenuate cardiac remodeling. Because of limited engraftment, most of the effects are possibly explained by paracrine activity of these cells.
• Ong, S., Lee, W., Huang, M., Dey, D., Kodo, K., Sanchez-Freire, V., Gold, J., & Wu, J. (2015). Response to letter regarding article, "Cross talk of combined gene and cell therapy in ischemic heart disease: Role of exosomal microRNA transfer". Circulation, 131(12). doi:10.1161/CIRCULATIONAHA.114.014467
• Ong, S., Lee, W., Huang, M., Dey, D., Kodo, K., Sanchez-Freire, V., Gold, J., & Wu, J. (2014). Cross talk of combined gene and cell therapy in ischemic heart disease role of exosomal MicroRNA transfer. Circulation, 130(11). doi:10.1161/CIRCULATIONAHA.113.007917
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  Background-Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs. Methods and Results-After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes. Conclusions-In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.
• Ong, S., Lee, W., Theodorou, L., Kodo, K., Lim, S., Shukla, D., Briston, T., Kiriakidis, S., Ashcroft, M., Davidson, S., Maxwell, P., Yellon, D., & Hausenloy, D. (2014). HIF-1 reduces ischaemia-reperfusion injury in the heart by targeting the mitochondrial permeability transition pore. Cardiovascular Research, 104(1). doi:10.1093/cvr/cvu172
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  Aims Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tolerance against acute myocardial ischaemia-reperfusion injury (IRI). However, the mechanism through which HIF-1 stabilization actually confers this cardioprotection is not clear. We investigated whether HIF-1α stabilization protects the heart against acute IRI by preventing the opening of the mitochondrial permeability transition pore (MPTP) and the potential mechanisms involved. Methods and results Stabilization of myocardial HIF-1 was achieved by pharmacological inhibition of prolyl hydroxylase (PHD) domain-containing enzyme using GSK360A or using cardiac-specific ablation of von Hippel-Lindau protein (VHLfl/fl) in mice. Treatment of HL-1 cardiac cells with GSK360A stabilized HIF-1, increased the expression of HIF-1 target genes pyruvate dehydrogenase kinase-1 (PDK1) and hexokinase II (HKII), and reprogrammed cell metabolism to aerobic glycolysis, thereby resulting in the production of less mitochondrial oxidative stress during IRI, and less MPTP opening, effects which were shown to be dependent on HKII. These findings were further confirmed when HIF-1 stabilization in the rat and murine heart resulted in smaller myocardial infarct sizes (both in vivo and ex vivo), decreased mitochondrial oxidative stress, and inhibited MPTP opening following IRI, effects which were also found to be dependent on HKII. Conclusions We have demonstrated that acute HIF-1α stabilization using either a pharmacological or genetic approach protected the heart against acute IRI by promoting aerobic glycolysis, decreasing mitochondrial oxidative stress, activating HKII, and inhibiting MPTP opening.
• Gu, M., Nguyen, P., Lee, A., Xu, D., Hu, S., Plews, J., Han, L., Huber, B., Lee, W., Gong, Y., De Almeida, P., Lyons, J., Ikeno, F., Pacharinsak, C., Connolly, A., Gambhir, S., Robbins, R., Longaker, M., & Wu, J. (2012). Microfluidic single-cell analysis shows that porcine induced pluripotent stem cell-derived endothelial cells improve myocardial function by paracrine activation. Circulation Research, 111(7). doi:10.1161/CIRCRESAHA.112.269001
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  RATIONALE: Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models. OBJECTIVE: To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia. METHODS AND RESULTS: Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P
• Lee, W., Warrington, J., Sonntag, W., & Lee, Y. (2012). Irradiation alters MMP-2/TIMP-2 system and collagen type IV degradation in brain. International Journal of Radiation Oncology Biology Physics, 82(5). doi:10.1016/j.ijrobp.2010.12.032
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  Purpose: Blood-brain barrier (BBB) disruption is one of the major consequences of radiation-induced normal tissue injury in the central nervous system. We examined the effects of whole-brain irradiation on matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in the brain. Methods and Materials: Animals received either whole-brain irradiation (a single dose of 10 Gy γ-rays or a fractionated dose of 40 Gy γ-rays, total) or sham-irradiation and were maintained for 4, 8, and 24 h following irradiation. mRNA expression levels of MMPs and TIMPs in the brain were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR). The functional activity of MMPs was measured by in situ zymography, and degradation of ECM was visualized by collagen type IV immunofluorescent staining. Results: A significant increase in mRNA expression levels of MMP-2, MMP-9, and TIMP-1 was observed in irradiated brains compared to that in sham-irradiated controls. In situ zymography revealed a strong gelatinolytic activity in the brain 24 h postirradiation, and the enhanced gelatinolytic activity mediated by irradiation was significantly attenuated in the presence of anti-MMP-2 antibody. A significant reduction in collagen type IV immunoreactivity was also detected in the brain at 24 h after irradiation. In contrast, the levels of collagen type IV were not significantly changed at 4 and 8 h after irradiation compared with the sham-irradiated controls. Conclusions: The present study demonstrates for the first time that radiation induces an imbalance between MMP-2 and TIMP-2 levels and suggests that degradation of collagen type IV, a major ECM component of BBB basement membrane, may have a role in the pathogenesis of brain injury. © 2012 Elsevier Inc.
• Conner, K., Forbes, M., Lee, W., Lee, Y., & Riddle, D. (2011). AT1 receptor antagonism does not influence early radiation-induced changes in microglial activation or neurogenesis in the normal rat brain. Radiation Research, 176(1). doi:10.1667/RR2560.1
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  Blockers of the renin-angiotensin-aldosterone system (RAAS) ameliorate cognitive deficits and some aspects of brain injury after whole-brain irradiation. We investigated whether treatment with the angiotensin II type 1 receptor antagonist L-158,809 at a dose that protects cognitive function after fractionated whole-brain irradiation reduced radiation-induced neuroinflammation and changes in hippocampal neurogenesis, well-characterized effects that are associated with radiation-induced brain injury. Male F344 rats received L-158,809 before, during and after a single 10-Gy dose of radiation. Expression of cytokines, angiotensin II receptors and angiotensin-converting enzyme 2 was evaluated by real-time PCR 24 h, 1 week and 12 weeks after irradiation. At the latter times, microglial density and proliferating and activated microglia were analyzed in the dentate gyrus of the hippocampus. Cell proliferation and neurogenesis were also quantified in the dentate subgranular zone. L-158,809 treatment modestly increased mRNA expression for Ang II receptors and TNF-α but had no effect on radiation-induced effects on hippocampal microglia or neurogenesis. Thus, although L-158,809 ameliorates cognitive deficits after whole-brain irradiation, the drug did not mitigate the neuroinflammatory microglial response or rescue neurogenesis. Additional studies are required to elucidate other mechanisms of normal tissue injury that may be modulated by RAAS blockers. © 2011 by Radiation Research Society.
• Lee, W., Cho, H., Sonntag, W., & Lee, Y. (2011). Radiation attenuates physiological angiogenesis by differential expression of VEGF, Ang-1, Tie-2 and Ang-2 in rat brain. Radiation Research, 176(6). doi:10.1667/RR2647.1
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  The etiology of radiation-induced cerebrovascular rarefaction remains unknown. In the present study, we examined the effect of whole-brain irradiation on endothelial cell (EC) proliferation/apoptosis and expression of various angiogenic factors in rat brain. F344×BN rats received either whole-brain irradiation (a single dose of 10 Gy γ rays) or sham irradiation and were maintained for 4, 8 and 24 h after irradiation. Double immunofluorescence staining was employed to visualize EC proliferation/apoptosis in brain. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), endothelial-specific receptor tyrosine kinase (Tie-2), and Ang-2 in brain were determined by real-time RT-PCR and immunofluorescence staining. A significant reduction in CD31-immunoreactive cells was detected in irradiated rat brains compared with sham-irradiated controls. Whole-brain irradiation significantly suppressed EC proliferation and increased EC apoptosis. In addition, a significant decrease in mRNA and protein expression of VEGF, Ang-1 and Tie-2 was observed in irradiated rat brains. In contrast, whole-brain irradiation significantly upregulated Ang-2 expression in rat brains. The present study provides novel evidence that whole-brain irradiation differentially affects mRNA and protein expression of VEGF, Ang-1, Tie-2 and Ang-2. These changes are closely associated with decreased EC proliferation and increased EC apoptosis in brain. © 2011 by Radiation Research Society.
• Lee, W., Sonntag, W., & Lee, Y. (2010). Aging attenuates radiation-induced expression of pro-inflammatory mediators in rat brain. Neuroscience Letter, 476(2). doi:10.1016/j.neulet.2010.04.009
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  The present study was designed to examine the effect of aging on radiation-induced expression of pro-inflammatory mediators in rat brain. Male F344 × BN rats (4, 16, and 24 months of age) received either whole brain irradiation with a single dose of 10. Gy γ-rays or sham-irradiation, and were maintained for 4, 8, and 24. h post-irradiation. The mRNA expression levels of various pro-inflammatory mediators such as cytokines, adhesion molecules, chemokine, and matrix metalloproteinase were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The acute inflammatory responses to irradiation, including overexpression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, monocyte chemoattractant protein-1 (MCP-1), and matrix metalloproteinase-9 (MMP-9) were markedly attenuated in the hippocampus of middle-aged and old rats compared with young groups. Specifically, a significant age-dependent decrease in TNF-α expression was detected 8 and 24. h after irradiation and a similar age-related attenuation was observed in IL-1β, ICAM-1, and VCAM-1 expression 4 and 8. h post-irradiation. MCP-1 expression was reduced 4. h post-irradiation and MMP-9 expression at 8. h post-irradiation. These results provide evidence for the first time that radiation-induced pro-inflammatory responses in the brain are suppressed in aged animals. © 2010 Elsevier Ireland Ltd.
• Lee, W., Sonntag, W., Mitschelen, M., Yan, H., & Lee, Y. (2010). Irradiation induces regionally specific alterations in pro-inflammatory environments in rat brain. International Journal of Radiation Biology, 86(2). doi:10.3109/09553000903419346
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  Purpose: Pro-inflammatory environments in the brain have been implicated in the onset and progression of neurological disorders. In the present study, we investigate the hypothesis that brain irradiation induces regionally specific alterations in cytokine gene and protein expression. Materials and methods: Four month old F344×BN rats received either whole brain irradiation with a single dose of 10 Gy γ-rays or sham-irradiation, and were maintained for 4, 8, and 24h following irradiation. The mRNA and protein expression levels of pro-inflammatory mediators were analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. To elucidate the molecular mechanisms of irradiation-induced brain inflammation, effects of irradiation on the DNA-binding activity of pro-inflammatory transcription factors were also examined. Results: A significant and marked up-regulation of mRNA and protein expression of pro-inflammatory mediators, including tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1), was observed in hippocampal and cortical regions isolated from irradiated brain. Cytokine expression was regionally specific since TNF-α levels were significantly elevated in cortex compared to hippocampus (57 greater) and IL-1β levels were elevated in hippocampus compared to cortical samples (126 greater). Increases in cytokine levels also were observed after irradiation of mouse BV-2 microglial cells. A series of electrophoretic mobility shift assays (EMSA) demonstrated that irradiation significantly increased activation of activator protein-1 (AP-1), nuclear factor-κB (NF-κB), and cAMP response element-binding protein (CREB). Conclusion: The present study demonstrated that whole brain irradiation induces regionally specific pro-inflammatory environments through activation of AP-1, NF-κB, and CREB and overexpression of TNF-α, IL-1β, and MCP-1 in rat brain and may contribute to unique pathways for the radiation-induced impairments in tissue function. © 2010 Informa UK Ltd.
• Lee, Y., Lee, W., & Kim, P. (2010). Oxidative mechanisms of IL-4-induced IL-6 expression in vascular endothelium. Cytokine, 49(1). doi:10.1016/j.cyto.2009.08.009
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  The present study is designed to investigate the effects of interleukin-4 (IL-4) on expression of interleukin-6 (IL-6), as well as to examine the role of distinct sources of reactive oxygen species (ROS) in this process. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that IL-4 significantly up-regulated the mRNA and protein expression of IL-6 in human aortic endothelial cells (HAEC) and C57BL/6 mice. Dihydroethidium (DHE) and dichlorofluorescein (DCF) fluorescence staining demonstrated that IL-4 significantly increased ROS generation in HAEC. A significant and dose-dependent inhibition of IL-4-induced IL-6 expression was observed in HAEC pre-treated with antioxidants, such as pyrrolidine dithiocarbamate (PDTC) and epigallocatechin gallate (EGCG), indicating that IL-4-induced IL-6 expression is mediated via an ROS-dependent mechanism. Additionally, pharmacological inhibitor of NADPH oxidase (NOX) significantly attenuated IL-4-induced ROS generation and IL-6 expression in HAEC. Furthermore, the disruption of NOX gene dramatically and significantly reduced IL-4-induced IL-6 expression in NOX knockout mice (B6.129S6-Cybbtm1Din/J). In contrast, overexpression of IL-6 in IL-4-activated HAEC was not affected by inhibiting other ROS generating pathways, such as xanthine oxidase, arachidonic acid metabolism, and the mitochondrial electron transport chain. These results demonstrate that IL-4 up-regulates IL-6 expression in vascular endothelium through NOX-mediated ROS generation. © 2009 Elsevier Ltd. All rights reserved.
• Lee, Y., Lee, W., & Kim, P. (2010). Role of NADPH oxidase in interleukin-4-induced monocyte chemoattractant protein-1 expression in vascular endothelium. Inflammation Research, 59(9). doi:10.1007/s00011-010-0187-3
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  Objective and design: The pro-oxidative and pro-inflammatory pathways in vascular endothelium have been implicated in the development of atherosclerosis. In the present study, we investigated effect of interleukin-4 (IL-4) on monocyte chemoattractant protein-1 (MCP-1) expression in vascular endothelium and examined the role of distinct sources of reactive oxygen species (ROS) in this process. Methods and results: Real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay showed that IL-4 significantly up-regulated mRNA and protein expression of MCP-1 in human aortic endothelial cells (HAEC) and C57BL/6 mice. A significant and dose-dependent inhibition of IL-4-induced MCP-1 expression was observed in HAEC pre-treated with antioxidants, such as pyrrolidine dithiocarbamate and epigallocatechin gallate, indicating that IL-4-induced MCP-1 expression is mediated via a ROS-dependent mechanism. Additionally, pharmacological inhibitors of NADPH oxidase (NOX) significantly attenuated IL-4-induced MCP-1 expression in HAEC. Furthermore, the disruption of the NOX gene dramatically reduced IL-4-induced MCP-1 expression in NOX knockout mice (B6.129S6-Cybbtm1Din/J). In contrast, overexpression of MCP-1 in IL-4-stimulated HAEC was not affected by inhibiting other ROS generating pathways, such as xanthine oxidase and the mitochondrial electron transport chain. Conclusions: These results demonstrate that IL-4 up-regulates MCP-1 expression in vascular endothelium through NOX-mediated ROS generation. © 2010 Springer Basel AG.
• Lee, W., Kang, S., Vlachos, P., & Lee, Y. (2009). A novel in vitro ischemia/reperfusion injury model. Archives of Pharmacal Research, 32(3). doi:10.1007/s12272-009-1316-9
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  The reperfusion of blood flow occurred in a number of conditions such as stroke and organ transplantation immensely augments tissue injury and causes more severe damage than prolonged ischemia. In the present study, we designed a novel double-layer parallel-plate flow chamber (PPFC) to develop an in vitro ischemia/reperfusion (I/R) injury model and examined the effects of I/R on inflammatory responses in human microvascular endothelial cells (HMEC-1). The expression of pro-inflammatory mediators, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1) in HMEC-1 was measured by quantitative real-time RT-PCR. The cells were also pre-treated with antioxidant pyrrolidine dithiocarbamate (PDTC) to verify involvement of an oxidative mechanism in I/R injury in vitro. The morphological changes and attenuated expression of pro-inflammatory mediators were observed in HMCE-1 exposed to the physiological flow. In contrast, I/R markedly and significantly up-regulated expression of pro-inflammatory mediators in HMEC-1. Additionally, pretreatment with PDTC significantly reduced I/R-mediated overexpression of pro-inflammatory mediators. The data from the present study provide evidence demonstrating that our newly designed PPFC can be utilized as an effective in vitro cell culture model system to develop new drugs specifically targeting against ischemia/reperfusion (I/R) injury. © 2009 The Pharmaceutical Society of Korea.
• Lee, W., & Lee, Y. (2008). Protective effects of genistein on proinflammatory pathways in human brain microvascular endothelial cells. Journal of Nutritional Biochemistry, 19(12). doi:10.1016/j.jnutbio.2007.10.006
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  Proinflammatory cerebromicrovascular environment has been implicated in the critical early pathologic events in a variety of neurodegenerative diseases. Recent studies also have demonstrated the potential beneficial effects of soy isoflavones. However, cellular and molecular mechanisms underlying these processes are not fully understood. The present study was designed to examine the hypothesis that soy isoflavone genistein may attenuate cytokine-induced proinflammatory pathways in human brain microvascular endothelial cells. The quantitative real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay showed that pretreatment of HBMEC with increasing concentrations of genistein significantly and dose-dependently inhibited cytokine-induced up-regulation of mRNA and protein expression of proinflammatory mediators such as tumor necrosis factor-α, interleukin-1β, monocyte chemoattractant protein-1, interleukin-8, and intercellular adhesion molecule-1. In addition, genistein pretreatment significantly reduced cytokine-mediated up-regulation of transmigration of blood leukocytes in a dose-dependent manner. Our results suggest that genistein may attenuate proinflammatory pathways through inhibition of cytokine-induced overexpression of proinflammatory mediators and inflammatory reactions in human brain microvascular endothelial cells. © 2008 Elsevier Inc. All rights reserved.