Ravi Goyal

Interests

Research

Mechanisms regulating angiogenesis;Gene Therapy;Epigenetic Regulation of Fetal Development

Teaching

Bioinformatics, Pharmacology, Physiology, Molecular Biology, Genetics and Epigenetics

Courses

Scholarly Contributions

Chapters

• Longo, L. D., & Goyal, R. (2014). Maternal Stress-Mediated Gene Expression Changes in the Mouse Placenta: A Mosaic Hypothesis of Epigenesis. In A Mosaic Hypothesis of Epigenetics. Academic Press. doi:10.1016/B978-0-12-394445-0.00037-0
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  Chapter Summary For optimal growth, development, maturation, and survival of the embryo/fetus, successful development and function of the placenta is crucial. Evidence from both epidemiologic studies in humans and experimental investigation in laboratory animals has demonstrated that antenatal maternal stress (protein/caloric deficiency or excess, hypoxia, and other invidious influences) establishes the foundation in utero for disease in the adult offspring. Here we consider aspects of gene expression and the role of epigenetics in the murine placenta as a consequence of maternal stressors. The mouse, a species in which the genome has been sequenced, and in which studies can be performed in a controlled manner, is a valuable “model” for such studies. Although each individual maternal stress is characterized by up- or downregulation of specific placental genes, functional analysis reveals some patterns of expression common to the several forms of stress. Of critical importance, these stress-altered genes include those involved in DNA methylation, histone modification, microRNA alteration, cell cycle regulation, and related global pathways of relevance to epigenetics in the developmental origins of adult health and disease. We attempt to synthesize the data and information into a coherent whole, presenting a mosaic hypothesis of epigenetic mechanisms to account for the various responses and their manifestations.

Journals/Publications

• Limesand, S. W., & Goyal, R. (2022). Epigenetic Modifications Guide Maturational Processes in Rat Pancreatic Islets. Endocrinology, 163(1).
• Goyal, R., & Limesand, S. W. (2021). Epigenetic Modifications Guide Maturational Processes in Rat Pancreatic Islets. Endocrinology, 163(1). doi:10.1210/endocr/bqab243
• Goyal, D., & Goyal, R. (2020). Angiogenic Transformation in Human Brain Micro Endothelial Cells: Whole Genome DNA Methylation and Transcriptomic Analysis. Frontiers in physiology, 10, 1502.
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  We tested the hypothesis that endothelial capillary tube formation in 3D cultures in basement membrane extract (BME) is secondary to the altered DNA promoter methylation and mRNA expression in human brain micro endothelial cells (HBMECs). We conducted a whole-genome transcriptomic and methylation microarray and CRISPR/Cas9-mediated gene knockdown to test our hypothesis. The data demonstrated that with angiogenic transformation 1318 and 1490 genes were significantly ( < 0.05) upregulated and downregulated, respectively. We compared our gene expression data with the published databases on GEO and found several genes in common. PTGS2, SELE, ID2, HSPA6, DLX2, HEY2, FOSB, SMAD6, SMAD7, and SMAD9 showed a very high level of expression during capillary tube formation. Among downregulated gene were ITGB4, TNNT1, PRSS35, TXNIP, IGFBP5. The most affected canonical pathways were ATM signaling and cell cycle G2/M DNA damage checkpoint regulation. The top upstream regulators of angiogenic transformation were identified to be VEGF, TP53, HGF, ESR1, and CDKN1A. We compared the changes in gene expression with the change in gene methylation and found hypomethylation of the CpG sites was associated with upregulation of 515 genes and hypermethylation was associated with the downregulation of 31 genes. Furthermore, the silencing of FOSB, FZD7, HEY2, HSPA6, NR4A3, SELE, PTGS2, SMAD6, SMAD7, and SMAD9 significantly inhibited angiogenic transformation as well as cell migration of HBMECs. We conclude that the angiogenic transformation is associated with altered DNA methylation and gene expression changes.
• Goyal, D., & Goyal, R. (2019). Angiogenic Transformation in Human Brain Micro Endothelial Cells: Whole Genome DNA Methylation and Transcriptomic Analysis.. Frontiers in physiology, 10, 1502. doi:10.3389/fphys.2019.01502
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  We tested the hypothesis that endothelial capillary tube formation in 3D cultures in basement membrane extract (BME) is secondary to the altered DNA promoter methylation and mRNA expression in human brain micro endothelial cells (HBMECs). We conducted a whole-genome transcriptomic and methylation microarray and CRISPR/Cas9-mediated gene knockdown to test our hypothesis. The data demonstrated that with angiogenic transformation 1318 and 1490 genes were significantly (p < 0.05) upregulated and downregulated, respectively. We compared our gene expression data with the published databases on GEO and found several genes in common. PTGS2, SELE, ID2, HSPA6, DLX2, HEY2, FOSB, SMAD6, SMAD7, and SMAD9 showed a very high level of expression during capillary tube formation. Among downregulated gene were ITGB4, TNNT1, PRSS35, TXNIP, IGFBP5. The most affected canonical pathways were ATM signaling and cell cycle G2/M DNA damage checkpoint regulation. The top upstream regulators of angiogenic transformation were identified to be VEGF, TP53, HGF, ESR1, and CDKN1A. We compared the changes in gene expression with the change in gene methylation and found hypomethylation of the CpG sites was associated with upregulation of 515 genes and hypermethylation was associated with the downregulation of 31 genes. Furthermore, the silencing of FOSB, FZD7, HEY2, HSPA6, NR4A3, SELE, PTGS2, SMAD6, SMAD7, and SMAD9 significantly inhibited angiogenic transformation as well as cell migration of HBMECs. We conclude that the angiogenic transformation is associated with altered DNA methylation and gene expression changes.
• Goyal, D., Limesand, S. W., & Goyal, R. (2019). Epigenetic responses and the developmental origins of health and disease. The Journal of endocrinology, 242(1), T105-T119.
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  Maternal and paternal factors influence offspring development and program its genome for successful postnatal life. Based on the stressors during gestation, the pregnant female prepares the fetus for the outside environment. This preparation is achieved by changing the epigenome of the fetus and is referred to as 'developmental programming'. For instance, nutritional insufficiency in utero will lead to programming events that prepare the fetus to cope up with nutrient scarcity following birth; however, offspring may not face nutrient scarcity following birth. This discrepancy between predicted and exposed postnatal environments are perceived as 'stress' by the offspring and may result in cardiovascular and metabolic disorders. Thus, this developmental programming may be both beneficial as well as harmful depending on the prenatal vs postnatal environment. Over the past three decades, accumulating evidence supports the hypothesis of Developmental Origin of Health and Disease (DOHaD) by the programming of the fetal phenotype without altering the genotype per se. These heritable modifications in gene expression occur through DNA methylation, histone modification and noncoding RNA-associated gene activation or silencing, and all are defined as epigenetic modifications. In the present review, we will summarize the evidence supporting epigenetic regulation as a significant component in DOHaD.
• Kar, S., Yadav, S. K., Goyal, R., Lefer, D. J., & Mishra, P. K. (2019). Abstract 433: Hydrogen Sulfide Protects the Heart Against Homocysteine-Induced Remodeling by Regulating Autophagy and Pyroptosis. Circulation Research, 125(Suppl_1). doi:10.1161/res.125.suppl_1.433
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  Hydrogen sulfide (H2S), a byproduct of homocysteine (Hcy) transsulfuration, protects against adverse cardiac remodeling in diabetic cardiomyopathy, hyperhomocysteinemia (HHcy), and other cardiomyop...
• Yates, D. T., Camacho, L. E., Kelly, A. C., Steyn, L. V., Davis, M. A., Antolic, A. T., Anderson, M. J., Goyal, R., Allen, R. E., Papas, K. K., Hay, W. W., & Limesand, S. W. (2019). Postnatal β2 adrenergic treatment improves insulin sensitivity in lambs with IUGR but not persistent defects in pancreatic islets or skeletal muscle. The Journal of physiology, 597(24), 5835-5858.
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  Previous studies in fetuses with intrauterine growth restriction (IUGR) have shown that adrenergic dysregulation was associated with low insulin concentrations and greater insulin sensitivity. Although whole-body glucose clearance is normal, 1-month-old lambs with IUGR at birth have higher rates of hindlimb glucose uptake, which may compensate for myocyte deficiencies in glucose oxidation. Impaired glucose-stimulated insulin secretion in IUGR lambs is due to lower intra-islet insulin availability and not from glucose sensing. We investigated adrenergic receptor (ADR) β2 desensitization by administering oral ADRβ modifiers for the first month after birth to activate ADRβ2 and antagonize ADRβ1/3. In IUGR lambs ADRβ2 activation increased whole-body glucose utilization rates and insulin sensitivity but had no effect on isolated islet or myocyte deficiencies. IUGR establishes risk for developing diabetes. In IUGR lambs we identified disparities in key aspects of glucose-stimulated insulin secretion and insulin-stimulated glucose oxidation, providing new insights into potential mechanisms for this risk.
• Ducsay, C. A., Goyal, R., Pearce, W. J., Wilson, S., Hu, X. Q., & Zhang, L. (2018). Gestational Hypoxia and Developmental Plasticity. Physiological reviews, 98(3), 1241-1334.
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  Hypoxia is one of the most common and severe challenges to the maintenance of homeostasis. Oxygen sensing is a property of all tissues, and the response to hypoxia is multidimensional involving complicated intracellular networks concerned with the transduction of hypoxia-induced responses. Of all the stresses to which the fetus and newborn infant are subjected, perhaps the most important and clinically relevant is that of hypoxia. Hypoxia during gestation impacts both the mother and fetal development through interactions with an individual's genetic traits acquired over multiple generations by natural selection and changes in gene expression patterns by altering the epigenetic code. Changes in the epigenome determine "genomic plasticity," i.e., the ability of genes to be differentially expressed according to environmental cues. The genomic plasticity defined by epigenomic mechanisms including DNA methylation, histone modifications, and noncoding RNAs during development is the mechanistic substrate for phenotypic programming that determines physiological response and risk for healthy or deleterious outcomes. This review explores the impact of gestational hypoxia on maternal health and fetal development, and epigenetic mechanisms of developmental plasticity with emphasis on the uteroplacental circulation, heart development, cerebral circulation, pulmonary development, and the hypothalamic-pituitary-adrenal axis and adipose tissue. The complex molecular and epigenetic interactions that may impact an individual's physiology and developmental programming of health and disease later in life are discussed.
• Goyal, D., & Goyal, R. (2018). Developmental Maturation and Alpha-1 Adrenergic Receptors-Mediated Gene Expression Changes in Ovine Middle Cerebral Arteries. Scientific reports, 8(1), 1772.
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  The Alpha Adrenergic Signaling Pathway is one of the chief regulators of cerebrovascular tone and cerebral blood flow (CBF), mediating its effects in the arteries through alpha1-adrenergic receptors (Alpha1AR). In the ovine middle cerebral artery (MCA), with development from a fetus to an adult, others and we have shown that Alpha1AR play a key role in contractile responses, vascular development, remodeling, and angiogenesis. Importantly, Alpha1AR play a significant role in CBF autoregulation, which is incompletely developed in a premature fetus as compared to a near-term fetus. However, the mechanistic pathways are not completely known. Thus, we tested the hypothesis that as a function of maturation and in response to Alpha1AR stimulation there is a differential gene expression in the ovine MCA. We conducted microarray analysis on transcripts from MCAs of premature fetuses (96-day), near-term fetuses (145-day), newborn lambs, and non-pregnant adult sheep (2-year) following stimulation of Alpha1AR with phenylephrine (a specific agonist). We observed several genes which belonged to pro-inflammatory and vascular development/angiogenesis pathway significantly altered in all of the four age groups. We also observed age-specific changes in gene expression-mediated by Alpha1AR stimulation in the different developmental age groups. These findings imply complex regulatory mechanisms of cerebrovascular development.
• Siebold, L., Obenaus, A., & Goyal, R. (2018). Criteria to define mild, moderate, and severe traumatic brain injury in the mouse controlled cortical impact model. Experimental neurology, 310, 48-57.
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  Traumatic brain injury (TBI) is a major health concern in the United States resulting in a substantial number of hospitalizations and in a broad spectrum of symptoms and disabilities. In the clinical setting, neurological responsiveness and structural imaging are used to classify mild, moderate and severe TBI. To evaluate the complex secondary and severity-specific injury response, investigators have relied on pre-clinical rodent models. The controlled cortical impact (CCI) model in mice is a widely used to study TBI. The CCI method has demonstrated consistent intra-laboratory outcomes due to precise control of cortical depth penetration, dwell time and speed of impact. While the CCI method results in control of injury severity, there is no consensus regarding the injury parameters or behavioral and histological endpoints that constitute a mild, moderate or severe TBI in this model. This discrepancy has resulted in considerable variability across laboratories in the outcomes of CCI-induced mild, moderate, and severe TBI. Inconsistent with clinical evaluation, injury severity in the CCI model has predominately relied on the extent of tissue damage. In the present review, we discuss variations in surgical parameters for injury induction as well as the criteria used to determine injury severity. Additionally, we propose guiding principles for the induction and defining of mild, moderate and severe TBI in the craniectomy-dependent experimental mouse CCI model.
• Goyal, R., & Clyman, R. I. (2017). Response to Coceani et al. Pediatric research, 82(2), 175.
• Kumar, M. M., & Goyal, R. (2017). LncRNA as a Therapeutic Target for Angiogenesis. Current topics in medicinal chemistry, 17(15), 1750-1757.
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  Out of 3 billion base pairs in human genome only ~2% code for proteins; and out of 180,000 transcripts in human cells, about 20,000 code for protein, remaining 160,000 are non-coding transcripts. Most of these transcripts are more than 200 base pairs and constitute a group of long non-coding RNA (lncRNA). Many of the lncRNA have its own promoter, and are well conserved in mammals. Accumulating evidence indicates that lncRNAs act as molecular switches in cellular differentiation, movement, apoptosis, and in the reprogramming of cell states by altering gene expression patterns. However, the role of this important group of molecules in angiogenesis is not well understood. Angiogenesis is a complex process and depends on precise regulation of gene expression.
• Mata-Greenwood, E., Goyal, D., & Goyal, R. (2017). Comparative and Experimental Studies on the Genes Altered by Chronic Hypoxia in Human Brain Microendothelial Cells. Frontiers in physiology, 8, 365.
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  Hypoxia inducible factor 1 alpha (HIF1A) is a master regulator of acute hypoxia; however, with chronic hypoxia, HIF1A levels return to the normoxic levels. Importantly, the genes that are involved in the cell survival and viability under chronic hypoxia are not known. Therefore, we tested the hypothesis that chronic hypoxia leads to the upregulation of a core group of genes with associated changes in the promoter DNA methylation that mediates the cell survival under hypoxia. We examined the effect of chronic hypoxia (3 days; 0.5% oxygen) on human brain micro endothelial cells (HBMEC) viability and apoptosis. Hypoxia caused a significant reduction in cell viability and an increase in apoptosis. Next, we examined chronic hypoxia associated changes in transcriptome and genome-wide promoter methylation. The data obtained was compared with 16 other microarray studies on chronic hypoxia. Nine genes were altered in response to chronic hypoxia in all 17 studies. Interestingly, HIF1A was not altered with chronic hypoxia in any of the studies. Furthermore, we compared our data to three other studies that identified HIF-responsive genes by various approaches. Only two genes were found to be HIF dependent. We silenced each of these 9 genes using CRISPR/Cas9 system. Downregulation of EGLN3 significantly increased the cell death under chronic hypoxia, whereas downregulation of ERO1L, ENO2, adrenomedullin, and spag4 reduced the cell death under hypoxia. We provide a core group of genes that regulates cellular acclimatization under chronic hypoxic stress, and most of them are HIF independent.
• Giang, M., Papamatheakis, D. G., Nguyen, D., Paez, R., Blum Johnston, C., Kim, J., Brunnell, A., Blood, Q., Goyal, R., Longo, L. D., & Wilson, S. M. (2016). Muscarinic Receptor Activation Affects Pulmonary Artery Contractility in Sheep: The Impact of Maturation and Chronic Hypoxia on Endothelium-Dependent and Endothelium-Independent Function. High altitude medicine & biology, 17(2), 122-32.
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  Giang, Michael, Demosthenes G. Papamatheakis, Dan Nguyen, Ricardo Paez, Carla Blum Johnston, Joon Kim, Alexander Brunnell, Quintin Blood, Ravi Goyal, Lawrence D. Longo, and Sean M. Wilson. Muscarinic receptor activation affects pulmonary artery contractility in sheep: the impact of maturation and chronic hypoxia on endothelium-dependent and endothelium-independent function. High Alt Med Biol. 17:122-132, 2015.-Muscarinic receptor activation in the pulmonary vasculature can cause endothelium-dependent vasodilation and smooth muscle-dependent vasoconstriction. Chronic hypoxia (CH) can modify both of these responses. This study aimed to assess the combined influence of CH and maturation on endothelium-dependent and endothelium-independent muscarinic-induced vasoreactivity. This was accomplished by performing wire myography on endothelium-intact or endothelium-disrupted pulmonary arterial rings isolated from normoxic or CH fetal and adult sheep. In endothelium-intact arteries, vasodilation was evaluated using cumulative bradykinin doses in phenylephrine and carbachol precontracted pulmonary arterial segments; and vasoconstriction was examined using cumulative doses of carbachol following bradykinin predilation. Effects of nonselective (atropine) and selective M1 (pirenzepine), M2 (AFDX116), and M3 (4-DAMP and Dau5884) muscarinic receptor antagonists were assessed in disrupted arteries. In normoxic arteries, bradykinin relaxation was twofold greater in the adult compared to fetus, while carbachol contraction was fourfold greater. In adult arteries, CH increased bradykinin relaxation and carbachol contraction. In vessels with intact endothelium, maturation and CH augmented maximal response and efficacy for carbachol constriction and bradykinin relaxation. Approximately 50%-80% of adult normoxic and CH endothelium-disrupted arteries contracted to acetylcholine, while ∼50% of fetal normoxic and ∼10% of CH arteries responded. Atropine reduced carbachol-induced contraction in all vessels. Adult normoxic vessels were most responsive to M3 antagonism, fetal to M2 antagonism, while M1 inhibition had no effect. Overall, muscarinic-induced pulmonary arterial contraction is partially endothelium dependent and appears to develop after birth. Fetuses are more reliant on M3 receptors while M2 receptors predominate in adults, whereas CH augments muscarinic-dependent pulmonary vasoconstriction in both.
• Goyal, R., Billings, T. L., Mansour, T., Martin, C., Baylink, D. J., Longo, L. D., Pearce, W. J., & Mata-Greenwood, E. (2016). Vitamin D status and metabolism in an ovine pregnancy model: effect of long-term, high-altitude hypoxia. American journal of physiology. Endocrinology and metabolism, 310(11), E1062-71.
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  Vitamin D status increases during healthy mammalian pregnancy, but the molecular determinants remain uncharacterized. The first objective of this study was to determine the effects of pregnancy, and the second objective was to examine the role of chronic hypoxia on vitamin D status and metabolism in an ovine model. We analyzed the plasma levels of cholecalciferol, 25-OH-D, and 1α,25-(OH)2D in nonpregnant ewes, near-term pregnant ewes, and their fetuses exposed to normoxia (low altitude) or hypoxia (high-altitude) for 100 days. Hypoxic sheep had increased circulating levels of 25-OH-D and 1α,25-(OH)2D compared with normoxic sheep. Hypoxia increases in 25-OH-D were associated with increased expression of renal 25-hydroxylases CYP2R1 and CYP2J. Pregnancy did not increase further the plasma levels of 25-OH-D, but it significantly increased those of the active metabolite, 1α,25-(OH)2D, in both normoxic and hypoxic ewes. Increased bioactivation of vitamin D correlated with increased expression of the vitamin D-activating enzyme CYP27b1 and decreased expression of the inactivating enzyme CYP24a1 in maternal kidneys and placentas. Hypoxia increased parathyroid hormone levels and further increased renal CYP27b1. Pregnancy and hypoxia decreased the expression of vitamin D receptor (VDR) in maternal kidney and lung, with opposite effects on placental VDR. We conclude that ovine pregnancy is a model of increased vitamin D status, and long-term hypoxia further improves vitamin D status due to pregnancy- and hypoxia-specific regulation of VDR and metabolic enzymes.
• Goyal, R., Goyal, D., Longo, L. D., & Clyman, R. I. (2016). Microarray gene expression analysis in ovine ductus arteriosus during fetal development and birth transition. Pediatric research, 80(4), 610-8.
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  Patent ductus arteriosus (PDA) in the newborn is the most common congenital heart anomaly and is significantly more common in preterm infants. Contemporary pharmacological treatment is effective in only 70-80% of the cases. Moreover, indomethacin or ibuprofen, which are used to close a PDA may be accompanied by serious side effects in premature infants. To explore the novel molecular pathways, which may be involved in the maturation and closure of the ductus arteriosus (DA), we used fetal and neonatal sheep to test the hypothesis that maturational development of DA is associated with significant alterations in specific mRNA expression.
• Dobyns, A. E., Goyal, R., Carpenter, L. G., Freeman, T. C., Longo, L. D., & Yellon, S. M. (2015). Macrophage gene expression associated with remodeling of the prepartum rat cervix: microarray and pathway analyses. PloS one, 10(3), e0119782.
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  As the critical gatekeeper for birth, prepartum remodeling of the cervix is associated with increased resident macrophages (Mφ), proinflammatory processes, and extracellular matrix degradation. This study tested the hypothesis that expression of genes unique to Mφs characterizes the prepartum from unremodeled nonpregnant cervix. Perfused cervix from prepartum day 21 postbreeding (D21) or nonpregnant (NP) rats, with or without Mφs, had RNA extracted and whole genome microarray analysis performed. By subtractive analyses, expression of 194 and 120 genes related to Mφs in the cervix from D21 rats were increased and decreased, respectively. In both D21 and NP groups, 158 and 57 Mφ genes were also more or less up- or down-regulated, respectively. Mφ gene expression patterns were most strongly correlated within groups and in 5 major clustering patterns. In the cervix from D21 rats, functional categories and canonical pathways of increased expression by Mφ gene related to extracellular matrix, cell proliferation, differentiation, as well as cell signaling. Pathways were characteristic of inflammation and wound healing, e.g., CD163, CD206, and CCR2. Signatures of only inflammation pathways, e.g., CSF1R, EMR1, and MMP12 were common to both D21 and NP groups. Thus, a novel and complex balance of Mφ genes and clusters differentiated the degraded extracellular matrix and cellular genomic activities in the cervix before birth from the unremodeled state. Predicted Mφ activities, pathways, and networks raise the possibility that expression patterns of specific genes characterize and promote prepartum remodeling of the cervix for parturition at term and with preterm labor.
• Goyal, R., & Longo, L. D. (2015). Metabolic Profiles in Ovine Carotid Arteries with Developmental Maturation and Long-Term Hypoxia. PloS one, 10(6), e0130739.
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  Long-term hypoxia (LTH) is an important stressor related to health and disease during development. At different time points from fetus to adult, we are exposed to hypoxic stress because of placental insufficiency, high-altitude residence, smoking, chronic anemia, pulmonary, and heart disorders, as well as cancers. Intrauterine hypoxia can lead to fetal growth restriction and long-term sequelae such as cognitive impairments, hypertension, cardiovascular disorders, diabetes, and schizophrenia. Similarly, prolonged hypoxic exposure during adult life can lead to acute mountain sickness, chronic fatigue, chronic headache, cognitive impairment, acute cerebral and/or pulmonary edema, and death.
• Goyal, R., Van-Wickle, J., Goyal, D., & Longo, L. D. (2015). Antenatal maternal low protein diet: ACE-2 in the mouse lung and sexually dimorphic programming of hypertension. BMC physiology, 15, 2.
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  Elevated blood pressure is an important global health problem, and in-utero under-nutrition may be an important factor in the pathogenesis of hypertension. In the present study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to sexually dimorphic developmental programming of the components of the pulmonary renin-angiotensin system. This may be important in the antenatal MLPD-associated development of hypertension. In pregnant mice, we administered normal (control) and isocaloric 50% protein restricted diet, commencing one week before mating and continuing until delivery of the pups. From the 18th to 24th week postnatal, we measured blood pressure in the offspring by use of a non-invasive tail-cuff method. In the same mice, we examined the mRNA and protein expression of the key components of the pulmonary renin-angiotensin system. Also, we examined microRNA complementary to angiotensin converting enzymes (ACE) 2 in the offspring lungs. Our results demonstrate that as a consequence of antenatal MLPD: 1) pup birthweight was significantly reduced in both sexes. 2) female offspring developed hypertension, but males did not. 3) In female offspring, ACE-2 protein expression was significantly reduced without any change in the mRNA levels. 4) miRNA 429, which has a binding site on ACE-2 - 3' UTR was significantly upregulated in the female antenatal MLPD offspring. 5) In males, ACE-2 mRNA and protein expression were unaltered. We conclude that in the mouse, antenatal MLPD-induced reduction of ACE-2 in the female offspring lung may be an important mechanisms in sexually dimorphic programming of hypertension.
• Jang, E. A., Longo, L. D., & Goyal, R. (2015). Antenatal maternal hypoxia: criterion for fetal growth restriction in rodents. Frontiers in physiology, 6, 176.
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  Rodents are a useful model for life science research. Accumulating evidence suggests that the offspring of mice and rats suffer from similar disorders as humans when exposed to hypoxia during pregnancy. Importantly, with antenatal hypoxic exposure, human neonates demonstrate low birth weight or growth restriction. Similarly, with antenatal hypoxic exposure rodents also demonstrate the fetal growth restriction (FGR). Surprisingly, there is no consensus on the minimum duration or degree of hypoxic exposure required to cause FGR in rodents. Thus, we have reviewed the available literature in an attempt to answer these questions. Based on studies in rats, birth weight reduction of 31% corresponded to 10th percentile reduction in birth weight curve. With the similar criterion (10th percentile), in mice 3 days or more and in rats 7 days or more of 14% or lower hypoxia administration was required to produce statistically significant FGR.
• Goyal, R., & Longo, L. D. (2014). Acclimatization to long-term hypoxia: gene expression in ovine carotid arteries. Physiological genomics, 46(19), 725-34.
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  Exposure to acute high-altitude hypoxia is associated with an increase in cerebral blood flow (CBF) as a consequence of low arterial O2 tension. However, in response to high altitude acclimatization, CBF returns to levels similar to those at sea level, and tissue blood flow is maintained by an increase in angiogenesis. Of consequence, dysregulation of the acclimatization responses and CBF can result in acute mountain sickness, acute cerebral and/or pulmonary edema. To elucidate the signal transduction pathways involved in successful acclimatization to high altitude, in ovine carotid arteries, we tested the hypothesis that high altitude-associated long-term hypoxia results in changes in gene expression of critical signaling pathways. We acclimatized nonpregnant adult sheep to 3,801 m altitude for ∼110 days and conducted oligonucleotide microarray experiments on carotid arteries. Of a total of 116 regulated genes, 58 genes were significantly upregulated and 58 genes were significantly downregulated (each >2-fold, P < 0.05). Major upregulated genes included suprabasin and myelin basic protein, whereas downregulated genes included BAG2. Several of these genes are known to activate the ERK canonical signal transduction pathway and the process of angiogenesis. We conclude that among other changes, the altered signal transduction molecules involved in high-altitude acclimatization are associated ERK activation and angiogenesis.
• Goyal, R., Goyal, D., Chu, N., Van Wickle, J., & Longo, L. D. (2014). Cerebral artery alpha-1 AR subtypes: high altitude long-term acclimatization responses. PloS one, 9(11), e112784.
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  In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P
• Goyal, R., Goyal, D., Wolfe, C., & Longo, L. D. (2014). Alpha1 adrenergic receptors and cerebral artery angiogenesis (679.4). The FASEB Journal, 28.
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  The largest neural component of the cerebral vasculature is adrenergic in nature. This serves an important role in regulating angiogenesis and maintaining adequate oxygenation to the brain tissue. Importantly, radio-ligand binding and molecular cloning in several species have demonstrated that the α1-adrenergic (α1-AR) family has three structurally distinct subtypes (α1A-, α1B-, α1D-) which are widely expressed in tissues including cerebral arteries. We tested the hypothesis that cerebral artery angiogenic responses are mediated by one of the three α1-AR subtypes. In an attempt to identify the specific α1-AR subtype, we examined their role and downstream signaling pathways in angiogenic responses in sheep cerebral arteries. In three dimensional organ culture experiments, we observed that the α1B-AR subtype (but not α1A- or α1D-AR subtypes) plays a critical role in angiogenesis responses. Also, these responses are mediated by canonical ERK-MAPK signaling pathways. We conclude that the development of specif...
• Goyal, R., Zhang, L., Blood, A. B., Baylink, D. J., Longo, L. D., Oshiro, B., & Mata-Greenwood, E. (2014). Characterization of an animal model of pregnancy-induced vitamin D deficiency due to metabolic gene dysregulation. American journal of physiology. Endocrinology and metabolism, 306(3), E256-66.
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  Vitamin D deficiency has been associated with pregnancy complications such as preeclampsia, gestational diabetes, and recurrent miscarriage. Therefore, we hypothesized differences in vitamin D status between healthy [Sprague-Dawley (SD) and Lewis (LW)] and complicated [Brown Norway (BN)] rat pregnancies. In SD, LW, and BN rats, we analyzed the maternal plasma levels of the vitamin D metabolites 25-OH-D and 1,25-(OH)2-D at prepregnancy, pregnancy, and postpartum. Analysis of the active metabolite 1,25-(OH)2-D showed a twofold increase in pregnant SD and LW rats but a nearly 10-fold decrease in pregnant BN rats compared with nonpregnant controls. BN rats had a pregnancy-dependent upregulation of CYP24a1 expression, a key enzyme that inactivates vitamin D metabolites. In contrast, the maternal renal expression of CYP24a1 in SD and LW rats remained constant throughout pregnancy. Analysis of the vitamin D receptor (VDR) indicated that LW and SD but not BN rats experience a pregnancy-induced 10-fold decrease in maternal renal VDR protein levels. Further analysis of bisulfite-converted and genomic DNA indicated that the observed differences in maternal renal regulation of CYP24a1 during pregnancy and lactation are not due to differences in CYP24a1 promoter methylation or single-nucleotide polymorphisms. Finally, supplementation with 1,25-(OH)2-D significantly improved the reproductive phenotype of BN rats by increasing litter size and maternal-fetal weight outcomes. We conclude that BN rats represent a novel animal model of pregnancy-specific vitamin D deficiency that is linked to pregnancy complications. Vitamin D deficiency in BN rats correlates with maternal renal CYP24a1 upregulation followed by CYP27b1 upregulation.
• Goyal, R., & Longo, L. D. (2013). Maternal protein deprivation: sexually dimorphic programming of hypertension in the mouse.. Hypertension research : official journal of the Japanese Society of Hypertension, 36(1), 29-35. doi:10.1038/hr.2012.129
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  Epidemiological data in humans and experiments in laboratory animals have demonstrated that the developmental programming of hypertension may occur as a consequence of dietary manipulations during pregnancy. Surprisingly, there is a scarcity of data regarding the development of hypertension as a consequence of a maternal low-protein diet (MLPD), particularly in the mouse. Furthermore, the role of sex in developmental programming is not well understood. We used FVB/NJ mice, because of their value in genetic/mechanistic analysis, to test the hypothesis that a MLPD during gestation leads to the sexually dimorphic developmental programming of hypertension and related disorders, such as intra-uterine growth restriction (IUGR), type 2 diabetes mellitus and obesity. We administered iso-caloric, normal (control), moderate protein (moderate MLPD) and severe protein (severe MLPD) diets to the mice, beginning 1 week before mating and continuing until the delivery of the pups. From 4 weeks onward, using a non-invasive tail-cuff method, we measured blood pressure and other parameters in the offspring. Our results demonstrate the following: (1) MLPD caused IUGR (low birthweight) in a dose-dependent manner; (2) Female offspring developed severe hypertension, whereas males were affected only moderately; (3) The blood glucose level was elevated only in females from the moderate MLPD group, although their insulin levels remained normal; (4) Rapid catch-up growth was observed in both sexes, with moderate MLPD females and severe MLPD males becoming overweight. Notably, blood leptin levels in the control group were significantly higher in females than in male offspring and were reduced in females from the severe MLPD group. We conclude that an antenatal MLPD during gestation leads to sexually dimorphic programming in mice.
• Goyal, R., Van Wickle, J., Goyal, D., Matei, N., & Longo, L. D. (2013). Antenatal maternal long-term hypoxia: acclimatization responses with altered gene expression in ovine fetal carotid arteries. PloS one, 8(12), e82200.
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  In humans and other species, long-term hypoxia (LTH) during pregnancy can lead to intrauterine growth restriction with reduced body/brain weight, dysregulation of cerebral blood flow (CBF), and other problems. To identify the signal transduction pathways and critical molecules, which may be involved in acclimatization to high altitude LTH, we conducted microarray with advanced bioinformatic analysis on carotid arteries (CA) from the normoxic near-term ovine fetus at sea-level and those acclimatized to high altitude for 110+ days during gestation. In response to LTH acclimatization, in fetal CA we identified mRNA from 38 genes upregulated >2 fold (P2-fold (P
• Goyal, R., Wong, C., Van Wickle, J., & Longo, L. D. (2013). Antenatal maternal protein deprivation: sexually dimorphic programming of the pancreatic renin-angiotensin system. Journal of the renin-angiotensin-aldosterone system : JRAAS, 14(2), 137-45.
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  As an underlying mechanism of antenatal maternal malnutrition-induced type 2 diabetes mellitus (T2DM), alterations in the local pancreatic renin-angiotensin system (RAS) may play a significant role. We tested the hypothesis that antenatal maternal protein deprivation (AMPD) leads to increased activity of the local pancreatic RAS, with associated hyperglycemia in the adult progeny. Mice dams were fed either control or 50% protein restricted diet (AMPD) starting one week before conception and maintained during complete gestation. Our results demonstrate low birth weight (control 1.5 ± 0.03 and AMPD 1.3 ± 0.03) and sexually dimorphic programming of the pancreatic RAS, with development of hyperglycemia only in the female mice offspring as a consequence of AMPD. No significant difference in serum insulin concentration was observed; however, AMPD was associated with increased mRNA and protein expression of angiotensinogen, renin and angiotensin-converting enzyme (ACE)-1 in male and female offspring. Of importance, mRNA and protein expression of ACE 2 and angiotensin II receptors was up-regulated only in the male offspring, as a consequence of AMPD. We conclude that sexually dimorphic programming of the pancreatic RAS expression is associated with AMPD diet-mediated development of hyperglycemia.
• Longo, L. D., & Goyal, R. (2013). Cerebral artery signal transduction mechanisms: developmental changes in dynamics and Ca2+ sensitivity. Current vascular pharmacology, 11(5), 655-711.
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  As compared to the adult, the developing fetus and newborn infant are at much greater risk for dysregulation of cerebral blood flow (CBF), with complications such as intraventricular and germinal matrix hemorrhage with resultant neurologic sequelae. To minimize this dysregulation and its consequences presents a major challenge. Although in many respects the fundamental signal transduction mechanisms that regulate relaxation and contraction pathways, and thus cerebrovascular tone and CBF in the immature organism are similar to those of the adult, the individual elements, pathways, and roles differ greatly. Here, we review aspects of these maturational changes of relaxation/contraction mechanisms in terms of both electro-mechanical and pharmaco-mechanical coupling, their biochemical pathways and signaling networks. In contrast to the adult cerebrovasculature, in addition to attenuated structure with differences in multiple cytoskeletal elements, developing cerebrovasculature of fetus and newborn differs in many respects, such as a strikingly increased sensitivity to [Ca(2+)]i and requirement for extracellular Ca(2+) for contraction. In essence, the immature cerebrovasculature demonstrates both "hyper-relaxation" and "hypo-contraction". A challenge is to unravel the manner in which these mechanisms are integrated, particularly in terms of both Ca(2+)-dependent and Ca(2+)-independent pathways to increase Ca(2+) sensitivity. Gaining an appreciation of these significant age-related differences in signal mechanisms also will be critical to understanding more completely the vulnerability of the developing cerebral vasculature to hypoxia and other stresses. Of vital importance, a more complete understanding of these mechanisms promises hope for improved strategies for therapeutic intervention and clinical management of intensive care of the premature newborn.
• Matei, N., Longo, L. D., & Goyal, R. (2013). High altitude long‐term hypoxia: Gene expression in adult ovine carotid arteries. The FASEB Journal, 27(S1). doi:10.1096/fasebj.27.1_supplement.1189.12
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  In humans and other species, exposure to long-term high altitude (>2500 m) is known to result in dysregulation of cerebral blood flow (CBF) as a consequence of low O2 tension. To elucidate the sign...
• Goyal, R., & Longo, L. D. (2012). Gene expression in sheep carotid arteries: major changes with maturational development. Pediatric research, 72(2), 137-46.
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  With development from immature fetus to near-term fetus, newborn, and adult, the cerebral vasculature undergoes a number of fundamental changes. Although the near-term fetus is prepared for a transition from an intra- to extra-uterine existence, this is not necessarily the case with the premature fetus, which is more susceptible to cerebrovascular dysregulation. In this study, we tested the hypothesis that the profound developmental and age-related differences in cerebral blood flow are associated with significant underlying changes in gene expression.
• Goyal, R., Henderson, D. A., Chu, N., & Longo, L. D. (2012). Ovine middle cerebral artery characterization and quantification of ultrastructure and other features: changes with development. American journal of physiology. Regulatory, integrative and comparative physiology, 302(4), R433-45.
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  Regulation of tone, blood pressure, and blood flow in the cerebral vasculature is of vital importance, particularly in the developing infant. We tested the hypothesis that, in addition to accretion of smooth muscle cells (SMCs) in cell layers with vessel thickening, significant changes in smooth muscle structure, as well as phenotype, extracellular matrix, and membrane proteins, in the media of cerebral arteries (CAs) during the course of late fetal development account for associated changes in contractility. Using transmission electron, confocal, wide-field epifluorescence, and light microscopy, we examined the structure and ultrastructure of CAs. Also, we utilized wire myography, Western immunoblotting, and real-time quantitative PCR to examine several other features of these arteries. We compared the main branch ovine middle CAs of 95- and 140-gestational day (GD) fetuses with those of adults (n = 5 for each experimental group). We observed a graded increase in phenylephrine- and KCl-induced contractile responses with development. Structurally, lumen diameter, media thickness, and media cross-sectional area increased dramatically from one age group to the next. With maturation, the cross-sectional profiles of CA SMCs changed from flattened bands in the 95-GD fetus to irregular ovoid-shaped fascicles in the 140-GD fetus and adult. We also observed a change in the type of collagen, specific integrin molecules, and several other parameters of SMC morphology with maturation. Ovine CAs at 95 GD appeared morphologically immature and poorly equipped to respond to major hemodynamic adjustments with maturation.
• Hartman, R. E., Kamper, J. E., Goyal, R., Stewart, J. M., & Longo, L. D. (2012). Motor and cognitive deficits in mice bred to have low or high blood pressure. Physiology & behavior, 105(4), 1092-7.
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  Deviations from normal blood pressure can lead to a number of physiological and behavioral complications. We tested the hypothesis that hyper- or hypotension is associated with significant differences in motor activity and coordination, anxiety levels, and spatial learning and memory in male and female mice. Compared to normotensive control mice, hypertensive mice were hyperactive and their performance was significantly worse on the rotarod (males only), cued learning (males only), spatial learning/re-learning, and spatial memory. Hypotensive mice of both genders swam more slowly and performed even worse than hypertensive mice on the rotarod, cued learning, spatial learning/re-learning, and spatial memory tasks. Across all phenotypes, females were generally more active than males in the open field and exhibited more anxiety-like behaviors in the elevated zero maze. Alterations in hemodynamics and/or neurovascular unit function may account for the observed behavioral changes in the hypo- and hypertensive mice.
• Goyal, R., Leitzke, A., Goyal, D., Gheorghe, C. P., & Longo, L. D. (2011). Antenatal maternal hypoxic stress: adaptations in fetal lung Renin-Angiotensin system. Reproductive sciences (Thousand Oaks, Calif.), 18(2), 180-9.
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  Antenatal maternal hypoxia (AMH) can lead to intrauterine growth restriction (IUGR), as well as idiopathic pulmonary hypertension of newborn and adult, the latter of which may be a consequence of alterations in the local pulmonary renin-angiotensin system (RAS). Little is known of these adaptations, however. Thus, we tested the hypothesis that antenatal maternal hypoxia is associated with alterations in gene and protein expression of the pulmonary renin-angiotensin system, which may play an important role in pulmonary disorders in the offspring. In FVB/NJ mice, we studied messenger RNA (mRNA) and protein expression, as well as promoter DNA methylation and microRNA (miRNA) levels in response to 48 hours hypoxia (10.5% O(2)) at 15.5 day post coitum (DPC). In response to AMH, the pulmonary mRNA levels of angiotensin-converting enzyme (ACE) 1.2, ACE-2, and angiotensin II type 1b (AT-1b) receptors were increased significantly, as compared to controls (N = 4). In response to antenatal hypoxia, pulmonary protein levels of renin and ACE-2 also were increased significantly, whereas ACE-1 protein expression was reduced. In fetal lungs, we also observed reduced expression of the miRNAs: mmu-mir -199b, -27b, -200b, and -468 that putatively increase the translation of renin, ACE-1, ACE-2, and AT-1 receptors, respectively. In response to AMH, promoter methylation of ACE was unchanged. We conclude that AMH leads to changes in expression of pulmonary RAS of fetal mice. The possible implications of these changes for the regulation of pulmonary vascular contractility in later life remain to be explored.
• Goyal, R., Lister, R., Leitzke, A., Goyal, D., Gheorghe, C. P., & Longo, L. D. (2011). Antenatal maternal hypoxic stress: adaptations of the placental renin-angiotensin system in the mouse. Placenta, 32(2), 134-9.
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  The stress of Antenatal Maternal Hypoxia (AMH) can lead to a number of physiological and pathological changes in both mother and fetus, changes which can be linked to alterations in placental morphology and gene regulation. Recently, in the Brown Norway rat "model" of placental insufficiency, we reported alterations in placental renin-angiotensin system (RAS) genes. Moreover, AMH can lead to reduced oxygen availability to the fetus, similar to a state of placental insufficiency. Thus, in pregnant mice dams we tested the hypothesis that antenatal maternal hypoxic stress leads to alterations in the placental RAS. These alterations may, in part, account for the phenotypic changes in both pregnant mice dams as well as fetus and adult offspring.
• Goyal, R., Papamatheakis, D. G., Loftin, M., Vrancken, K., Dawson, A. S., Osman, N. J., Blood, A. B., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2011). Long-term maternal hypoxia: the role of extracellular Ca2+ entry during serotonin-mediated contractility in fetal ovine pulmonary arteries. Reproductive sciences (Thousand Oaks, Calif.), 18(10), 948-62.
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  Antenatal maternal long-term hypoxia (LTH) can alter serotonin (5-HT) and calcium (Ca(2+)) signaling in fetal pulmonary arteries (PAs) and is associated with persistent pulmonary hypertension of the newborn (PPHN). In humans, the antenatal maternal hypoxia can be secondary to smoking, anemia, and chronic obstructive pulmonary disorders. However, the mechanisms of antenatal maternal hypoxia-related PPHN are unresolved. Because both LTH and 5-HT are associated with PPHN, we tested the hypothesis that antenatal maternal LTH can increase 5-HT-mediated PA contraction and associated extracellular Ca(2+) influx through L-type Ca(2+) channels (Ca(L)), nonselective cation channels (NSCCs), and reverse-mode sodium-calcium exchanger (NCX) in the near-term fetus. We performed wire myography and confocal-Ca(2+) imaging approaches on fetal lamb PA (∼ 140 days of gestation) from normoxic ewes or those acclimatized to high-altitude LTH (3801 m) for ∼110 days. Long-term hypoxia reduced the potency but not the efficacy of 5-HT-induced PA contraction. Ketanserin (100 nmol/L), a 5-HT(2A) antagonist, shifted 5-HT potency irrespective of LTH, while GR-55562 (1 µmol/L), a 5-HT(1B/D) inhibitor, antagonized 5-HT-induced contraction in normoxic fetuses only. Various inhibitors for Ca(L), NSCC, and reverse-mode NCX were used in contraction studies. Contraction was reliant on extracellular Ca(2+) regardless of maternal hypoxia, NSCC was more important to contraction than Ca(L), and reverse-mode NCX had little or no role in contraction. Long-term hypoxia also attenuated the effects of 2-APB and flufenamic acid and reduced Ca(2+) responses observed by imaging studies. Overall, LTH reduced 5HT(1B/D) function and increased NSCC-related Ca(2+)-dependent contraction in ovine fetuses, which may compromise pulmonary vascular function in the newborn.
• Papamatheakis, D. G., Vemulakonda, S., Blood, Q., Goyal, R., Rubalcava, M., Vrancken, K., Bennett, A., Dawson, A., Osman, N. J., Blood, A. B., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2011). Preservation of serotonin-mediated contractility in adult sheep pulmonary arteries following long-term high-altitude hypoxia. High altitude medicine & biology, 12(3), 253-64.
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  Long-term hypoxia (LTH) can increase serotonin (5-HT) signaling as well as extracellular calcium entry in adult rodent pulmonary arteries (PA), and 5-HT is associated with pulmonary hypertension. Because LTH, 5-HT, and calcium entry are related, we tested the hypothesis that LTH increases 5-HT-mediated PA contractility and associated calcium influx through L-type Ca2+ channels, nonselective cation channels (NSCC), and reverse-mode sodium-Ca2+ exchange. We performed wire myography and confocal calcium imaging on pulmonary arteries from adult ewes that lived near sea level or were maintained at high-altitude (3801 m) for ∼110 days. LTH did not increase the arterial medial wall thickness, nor did it affect the potency or efficacy for 5-HT-induced PA contraction. Ketanserin (100 nM), a 5-HT2A antagonist, shifted the 5-HT potency to a far greater extent than 1 μM GR-55562, a 5-HT1B/D inhibitor. These influences were unaffected by LTH. The rank order for reducing 5-HT-induced PA contraction in normoxic animals was extracellular calcium removal≈10 mM Ni2+≈10 μM verapamil≈10 μM nifedipine with 50 μM SKF 96365>30 μM KB-R7943≈100 μM flufenamic acid≈10 μM nifedipine≈100 μM Gd3+> 100 μM La3+>500 μM Ni2+≈10 μM diltiazem≈50 μM 2-APB≈100 μM LOE 908. Contraction was not reduced by 100 μM spermine or 30 μM SN-6. LTH increased the effects of KB-R7943 and mitigated those of nifedipine but did not affect calcium responses in imaging studies. Overall, in adult sheep, arterial structure and 5-HT2A and 5HT1B/D functions are preserved following LTH while the role of NSCC-related calcium-dependent contraction is increased. These elements indicate preservation of PA contractility in LTH with minimal functional changes.
• Chu, N., Goyal, R., Goyal, D., & Longo, L. D. (2010). Maturation and Differential Role of PKC Isoforms in Adrenergic, Serotonergic, and Phorbol Ester-Mediated Cerebral Artery Contractility. The FASEB Journal, 24.
• Gheorghe, C. P., Goyal, R., Mittal, A., & Longo, L. D. (2010). Gene expression in the placenta: maternal stress and epigenetic responses. The International journal of developmental biology, 54(2-3), 507-23.
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  Successful placental development is crucial for optimal growth, development, maturation and survival of the embryo/fetus into adulthood. Numerous epidemiologic and experimental studies have demonstrated the profound influence of intrauterine environment on life, and the diseases to which one is subject as an adult. For the most part, these invidious influences, whether maternal hypoxia, protein or caloric deficiency or excess, and others, represent types of maternal stress. In the present review, we examine certain aspects of gene expression in the placenta as a consequence of maternal stressors. To examine these issues in a controlled manner, and in a species in which the genome has been sequenced, most of these reported studies have been performed in the mouse. Although each individual maternal stress is characterized by up- or down-regulation of specific genes in the placenta, functional analysis reveals some patterns of gene expression common to the several forms of stress. Of critical importance, these genes include those involved in DNA methylation and histone modification, cell cycle regulation, and related global pathways of great relevance to epigenesis and the developmental origins of adult health and disease.
• Goyal, R., Chu, N., Goyal, D., & Longo, L. D. (2010). Organ Culture: Effect on Fetal and Adult Cerebral Arterial Contractility. The FASEB Journal, 24.
• Goyal, R., Goyal, D., Leitzke, A., Gheorghe, C. P., & Longo, L. D. (2010). Brain renin-angiotensin system: fetal epigenetic programming by maternal protein restriction during pregnancy. Reproductive sciences (Thousand Oaks, Calif.), 17(3), 227-38.
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  Maternal protein malnutrition during pregnancy can lead to significant alterations in the systemic renin-angiotensin system (RAS) in the fetus. All components of the RAS are present in brain and may be altered in many disease states. Importantly, these disorders are reported to be of higher incidence in prenatally malnourished individuals. In the current study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to epigenetic changes and alterations in gene expression of brain RAS of the mouse fetus.
• Goyal, R., Leitzke, A., Goyal, D., & Longo, L. D. (2010). Antenatal Protein Deprivation in the Mouse: Epigenetic Changes and Developmental Origin of Hypertension in Adult. The FASEB Journal, 24.
• Goyal, R., Leitzke, A., Goyal, D., Gheorghe, C. P., & Longo, L. D. (2010). Antenatal Maternal Hypoxic Stress: Epigenetic Adaptations in Fetal Lung Renin-Angiotensin System. The FASEB Journal, 24.
• Goyal, R., Mittal, A., Chu, N., Arthur, R. A., Zhang, L., & Longo, L. D. (2010). Maturation and long-term hypoxia-induced acclimatization responses in PKC-mediated signaling pathways in ovine cerebral arterial contractility. American journal of physiology. Regulatory, integrative and comparative physiology, 299(5), R1377-86.
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  In the developing fetus, cerebral arteries (CA) show striking differences in signal transduction mechanisms compared with the adult, and these differences are magnified in response to high-altitude long-term hypoxia (LTH). In addition, in the mature organism, cerebrovascular acclimatization to LTH may be associated with several clinical problems, the mechanisms of which are unknown. Because PKC plays a key role in regulating CA contractility, in fetal and adult cerebral arteries, we tested the hypothesis that LTH differentially regulates the PKC-mediated Ca(2+) sensitization pathways and contractility. In four groups of sheep [fetal normoxic (FN), fetal hypoxic (FH), adult normoxic (AN), and adult hypoxic (AH)], we examined, simultaneously, responses of CA tension and intracellular Ca(2+) concentration and measured CA levels of PKC, ERK1/2, RhoA, 20-kDa myosin light chain, and the 17-kDa PKC-potentiated myosin phosphatase inhibitor CPI-17. The PKC activator phorbol 12,13-dibutyrate (PDBu) produced robust contractions in all four groups. However, PDBu-induced contractions were significantly greater in AH CA than in the other groups. In all CA groups except AH, in the presence of MEK inhibitor (U-0126), the PDBu-induced contractions were increased a further 20-30%. Furthermore, in adult CA, PDBu led to increased phosphorylation of ERK1, but not ERK2; in fetal CA, the reverse was the case. PDBu-stimulated ERK2 phosphorylation also was significantly greater in FH than FN CA. Also, although RhoA/Rho kinase played a significant role in PDBu-mediated contractions of FN CA, this was not the case in FH or either adult group. Also, whereas CPI-17 had a significant role in adult CA contractility, this was not the case for the fetus. Overall, in ovine CA, the present study demonstrates several important maturational and LTH acclimatization changes in PKC-induced contractile responses and downstream pathways. The latter may play a key role in the pathophysiologic disorders associated with acclimatization to high altitude.
• Goyal, R., Mittal, A., Chu, N., Zhang, L., & Longo, L. D. (2010). alpha(1)-Adrenergic receptor subtype function in fetal and adult cerebral arteries. American journal of physiology. Heart and circulatory physiology, 298(6), H1797-806.
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  In the developing fetus, cerebral artery (CA) contractility demonstrates significant functional differences from that of the adult. This may be a consequence of differential activities of alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes. Thus we tested the hypothesis that maturational differences in adrenergic-mediated CA contractility are, in part, a consequence of differential expression and/or activities of alpha(1)-AR subtypes. In CA from fetal ( approximately 140 days) and nonpregnant adult sheep, we used wire myography and imaging, with simultaneous measurement of tension and intracellular Ca(2+) concentration ([Ca(2+)](i)), radioimmunoassay, and Western immunoblots to examine phenylephrine (Phe)-induced contractile responses. The alpha(1A)-AR antagonists (5-MU and WB-4101) completely inhibited Phe-induced contraction in adult but not fetal CA; however, [Ca(2+)](i) increase was reduced significantly in both age groups. The alpha(1D)-AR antagonist (BMY-7378) blocked both Phe-induced contractions and Ca(2+) responses to a significantly greater extent in adult compared with fetal CA. In both age groups, inhibition of alpha(1A)-AR and alpha(1B)-AR, but not alpha(1D)-AR, significantly reduced inositol 1,4,5-trisphosphate responses to Phe. Western immunoblots demonstrated that the alpha(1)-AR subtype expression was only approximately 20% in fetal CA compared with the adult. Moreover, in fetal CA, the alpha(1D)-AR was expressed significantly greater than the other two subtypes. Also, in fetal but not adult CA, Phe induced a significant increase in activated ERK1/2; this increase in phosphorylated ERK was blocked by alpha(1B)-AR (CEC) and alpha(1D)-AR (BMY-7378) inhibitors, but not by alpha(1A)-AR inhibitors (5-MU or WB-4101). In conclusion, in the fetal CA, alpha(1B)-AR and alpha(1D)-AR subtypes play a key role in contractile response as well as in ERK activation. We speculate that in fetal CA alpha(1B)-AR and alpha(1D)-AR subtypes may be a critical factor associated with cerebrovascular growth and function.
• Goyal, R., Yellon, S. M., Longo, L. D., & Mata-Greenwood, E. (2010). Placental gene expression in a rat 'model' of placental insufficiency. Placenta, 31(7), 568-75.
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  Placental insufficiency is a major factor associated with pregnancy complications such as miscarriages, intrauterine growth restriction and pre-eclampsia. Recent studies have identified the Brown Norway (BN) rat as a natural 'model' of placental insufficiency associated with decreased trophoblast remodeling of maternal uterine arteries.
• Gheorghe, C. P., Goyal, R., Holweger, J. D., & Longo, L. D. (2009). Placental gene expression responses to maternal protein restriction in the mouse. Placenta, 30(5), 411-7.
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  Maternal protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. We tested the hypothesis that, by the use of microarray technology, we could identify specific genes and cellular pathways in the developing placenta that are responsive to maternal protein deprivation, and propose a potential mechanism for observed gene expression changes.
• Goyal, R., Angermann, J. E., Ostrovskaya, O., Buchholz, J. N., Smith, G. D., & Wilson, S. M. (2009). Enhanced capacitative calcium entry and sarcoplasmic-reticulum calcium storage capacity with advanced age in murine mesenteric arterial smooth muscle cells. Experimental gerontology, 44(3), 201-7.
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  Intracellular Ca(2+) signaling is important to perfusion pressure related arterial reactivity and to vascular disorders including hypertension, angina and ischemic stroke. We have recently shown that advancing-age leads to calcium signaling adaptations in mesenteric arterial myocytes from C57 BL/6 mice [Corsso, C.D., Ostrovskaya, O., McAllister, C.E., Murray, K., Hatton, W.J., Gurney, A.M., Spencer, N.J., Wilson, S.M., 2006. Effects of aging on Ca(2+) signaling in murine mesenteric arterial smooth muscle cells. Mech. Ageing Dev. 127, 315-323)] which may contribute to decrements in perfusion pressure related arterial contractility others have shown occur. Even still, the mechanisms underlying the changes in Ca(2+) signaling and arterial reactivity are unresolved. Ca(2+) transport and storage capabilities are thought to contribute to age-related Ca(2+) signaling dysfunctions in other cell types. The present studies were therefore designed to test the hypothesis that cytosolic and compartmental Ca(2+) homeostasis in mesenteric arterial myocytes changes with advanced age. The hypothesis was tested by performing digitalized fluorescence microscopy on mesenteric arterial myocytes isolated from 5- to 6-month and 29- to 30-month-old C57Bl/6 mice. The data provide evidence that with advanced age capacitative Ca(2+) entry and sarcoplasmic reticulum Ca(2+) storage are increased although sarcoplasmic reticulum Ca(2+) uptake and plasma membrane Ca(2+) extrusion are unaltered. Overall, the studies begin to resolve the mechanisms associated with age-related alterations in mesenteric arterial smooth muscle Ca(2+) signaling and their physiological consequences.
• Goyal, R., Galffy, A., Field, S. A., Gheorghe, C. P., Mittal, A., & Longo, L. D. (2009). Maternal protein deprivation: changes in systemic renin-angiotensin system of the mouse fetus.. Reproductive sciences (Thousand Oaks, Calif.), 16(9), 894-904. doi:10.1177/1933719109337260
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  We tested the hypothesis that maternal protein deprivation during gestation results in changes in expression of the systemic renin-angiotensin system in fetal mice. Fetal weight was decreased significantly as a consequence of 50% maternal protein deprivation during second half of gestation. In fetal liver, angiotensinogen protein expression was reduced significantly despite a significant increase in messenger RNA (mRNA). In fetal kidneys, both mRNA and protein levels of renin were increased significantly. In the lungs, we observed a decrease in both angiotensin-converting enzyme I and II mRNA expression, whereas protein expression of both isoforms was increased significantly. The fetal heart showed significant increases in expression of angiotensin II type 1 (AT-1) and type 2 (AT-2) receptors mRNA. Protein expression of AT-1 receptors increased, while that of AT-2 receptors decreased. We conclude that maternal low-protein diet during gestation leads to significant changes in expression of the systemic renin-angiotensin system in fetal mice and may be important in the genesis of hypertension in the adult.
• Goyal, R., Leitzke, A., Goyal, D., & Longo, L. D. (2009). Maternal low protein diet: Developmental origin of adult hypertension. The FASEB Journal, 23.
• Goyal, R., Mittal, A., Chu, N., Shi, L., Zhang, L., & Longo, L. D. (2009). Maturation and the role of PKC-mediated contractility in ovine cerebral arteries. American journal of physiology. Heart and circulatory physiology, 297(6), H2242-52.
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  Ca2+-independent pathways such as protein kinase C (PKC), extracellular-regulated kinases 1 and 2 (ERK1/2), and Rho kinase 1 and 2 (ROCK1/2) play important roles in modulating cerebral vascular tone. Because the roles of these kinases vary with maturational age, we tested the hypothesis that PKC differentially regulates the Ca2+-independent pathways and their effects on cerebral arterial contractility with development. We simultaneously examined the responses of arterial tension and intracellular Ca2+ concentration and used Western immunoblot analysis to measure ERK1/2, RhoA, 20 kDa regulatory myosin light chain (MLC20), PKC-potentiated inhibitory protein of 17 kDa (CPI-17), and caldesmon. Phorbol 12,13-dibutyrate (PDBu)-mediated PKC activation produced a robust contractile response, which was increased a further 20 to 30% by U-0126 (MEK inhibitor) in cerebral arteries of both age groups. Of interest, in the fetal cerebral arteries, PDBu leads to an increased phosphorylation of ERK2 compared with ERK1, whereas in adult arteries, we observed an increased phosphorylation of ERK1 compared with ERK2. Also, in the present study, RhoA/ROCK played a significant role in the PDBu-mediated contractility of fetal cerebral arteries, whereas in adult cerebral arteries, CPI-17 and caldesmon had a significantly greater role compared with the fetus. PDBu also led to an increased MLC20 phosphorylation, a response blunted by the inhibition of myosin light chain kinase only in the fetus. Overall, the present study demonstrates an important maturational shift from RhoA/ROCK-mediated to CPI-17/caldesmon-mediated PKC-induced contractile response in ovine cerebral arteries.
• Goyal, R., Mittal, A., Shi, L., Wedhas, N., & Longo, L. D. (2009). High altitude acclimatization responses in cerebral artery PKC-dependent signaling pathway proteins: Changes with development. The FASEB Journal, 23.
• Goyal, R., Vemulakonda, S., Mittal, A., Loftin, M., Vranken, K., Nguyen, D., Blood, A. B., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2009). Chronic hypoxia influences the role of extracellular Ca2+ influx during serotonin-mediated contractility of pulmonary arteries from adult sheep. The FASEB Journal, 23.
• Mccrate, B., Webster, M., Osman, N., Nguyen, D., Goyal, R., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2009). Changes in pulmonary arterial smooth muscle structure with maturation and chronic hypoxia in sheep. The FASEB Journal, 23.
• Mittal, A., Goyal, R., Shi, L., Wedhas, N., & Longo, L. D. (2009). Role of ERK1/2 and Rho A/Rho kinase in PKC-induced contractions in ovine cerebral arteries: A developmental study. The FASEB Journal, 23.
• Mittal, A., Goyal, R., Wedhas, N., Shi, L., & Longo, L. D. (2009). Differential role of {alpha}1-adrenergic receptor subtypes in Ca2+-dependent and Ca2+-independent contraction in developing cerebral arteries. The FASEB Journal, 23.
• Wilson, S. M., Webster, M., Vranken, K., Vemulakonda, S., Pearce, W. J., Nguyen, D., Mittal, A., Longo, L. D., Loftin, M., Goyal, R., & Blood, A. B. (2009). Maternal chronic hypoxic stress influences the role of extracellular Ca2+ influx to serotonin-mediated contractility of pulmonary arteries from fetal sheep. The FASEB Journal, 23(S1). doi:10.1096/fasebj.23.1_supplement.619.4
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  Cytosolic Ca2+ is central to pulmonary vascular reactivity and important to pathogenesis due to hypoxic stress, but the role of Ca2+ to disease in the unborn fetus is not well understood. We theref...
• Goyal, R., Creel, K. D., Chavis, E., Smith, G. D., Longo, L. D., & Wilson, S. M. (2008). Effects of maturation on intracellular Ca2+ homeostasis in ovine pulmonary arterial smooth muscle cells. The FASEB Journal, 22.
• Goyal, R., Creel, K. D., Chavis, E., Smith, G. D., Longo, L. D., & Wilson, S. M. (2008). Maturation of intracellular calcium homeostasis in sheep pulmonary arterial smooth muscle cells. American journal of physiology. Lung cellular and molecular physiology, 295(5), L905-14.
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  Cytosolic Ca(2+) signaling dynamics are important to pulmonary arterial reactivity, and alterations are implicated in pulmonary vascular disorders. Yet, adaptations in cellular Ca(2+) homeostasis and receptor-mediated Ca(2+) signaling with maturation from fetal to adult life in pulmonary arterial smooth muscle cells (PASMCs) are not known. The present study tested the hypothesis that cytosolic Ca(2+) homeostasis and receptor-generated Ca(2+) signaling adapt with maturation in sheep PASMCs. Digitalized fluorescence microscopy was performed using isolated PASMCs from fetal and adult sheep that were loaded with the Ca(2+) indicator fura 2. The results show that basal cytosolic and sarcoplasmic reticulum Ca(2+) levels are attained before birth. Similarly, Ca(2+) efflux pathways from the cytosol and basal as well as capacitative Ca(2+) entry (CCE) are also developed before birth. However, receptor-mediated Ca(2+) signaling adapts with maturation. Prominently, serotonin stimulation elicited Ca(2+) elevations in very few fetal compared with adult PASMCs; in contrast, phenylephrine elevated Ca(2+) in a similar percentage of fetal and adult PASMCs. Serotonin and phenylephrine elicited Ca(2+) increases of a similar magnitude in reactive cells of fetus and adult, supporting the assertion that inositol trisphosphate signaling is intact. Caffeine and ATP elevated Ca(2+) in equivalent numbers of fetal and adult PASMCs. However, the caffeine-induced cytosolic Ca(2+) increase was significantly greater in fetal PASMCs, whereas the ATP-elicited increase was greater in adult cells. Overall, the results of this study demonstrate selective adaptations in receptor-mediated Ca(2+) signaling, but not in cellular Ca(2+) homeostasis.
• Goyal, R., Loftin, M., Derrick, A., Nguyen, D., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2008). 5-HT2A receptor mediated contractility of Ovine pulmonary arteries: Effects of maturation and chronic hypoxia. The FASEB Journal, 22.
• Goyal, R., Loftin, M., Derrick, A., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2008). Role of calcium to serotonergic mediated contractility in ovine pulmonary arteries: effects of maturation and chronic hypoxia. The FASEB Journal, 22.
• Goyal, R., Nguyen, D., Loftin, M., Derrick, A., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2008). Contributions of PKC, RhoA and ERK signaling to serotonergic contractility of pulmonary arteries from chronic hypoxic fetal and adult sheep. The FASEB Journal, 22.
• Goyal, R., Nguyen, D., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2008). Role of reverse-mode sodium-calcium exchange to serotonergic contractility in pulmonary arteries of hypoxic sheep. The FASEB Journal, 22.
• Nguyen, D., Goyal, R., Derrick, A., Loftin, M., Osman, N., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2008). Acetylcholine receptor-mediated contractility of ovine pulmonary arteries: Changes with maturation and chronic hypoxia. The FASEB Journal, 22.
• Goyal, R., Pearce, W. J., Longo, L. D., & Wilson, S. M. (2007). Chronic hypoxia and the influence of maturation on serotonergic contractility in Ovine pulmonary arteries. The FASEB Journal, 21(6).
• Ostrovskaya, O., Goyal, R., Osman, N., McAllister, C. E., Pessah, I. N., Hume, J. R., & Wilson, S. M. (2007). Inhibition of ryanodine receptors by 4-(2-aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) in canine pulmonary arterial smooth muscle cells. The Journal of pharmacology and experimental therapeutics, 323(1), 381-90.
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  Ryanodine is a selective ryanodine receptor (RyR) blocker, with binding dependent on RyR opening. In whole-cell studies, ryanodine binding can lock the RyR in an open-conductance state, short-circuiting the sarcoplasmic reticulum, which restricts studies of inositol-1,4,5-trisphosphate receptor (InsP3R) activity. Other RyR blockers have nonselective effects that also limit their utility. 4-(2-aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) blocks RyR-elicited Ca2+ increases in skeletal and cardiac muscle; yet, its actions on smooth muscle are unknown. Canine pulmonary arterial smooth muscle cells (PASMCs) express both RyRs and InsP3Rs; thus, we tested the ability of FLA 365 to block RyR- and serotonin-mediated InsP3R-elicited Ca2+ release by imaging fura-2-loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca2+ increases that were reversibly reduced by FLA 365, with an estimated IC50 of approximately 1 to 1.5 microM, and inhibited by 10 microM ryanodine or 10 microM cyclopiazonic acid. FLA 365 also blocked L-type Ca2+ channel activity, with 10 microM reducing Ba2+ current amplitude in patch voltage-clamp studies to 54 +/- 6% of control and 100 microM FLA 365 reducing membrane current to 21 +/- 6%. InsP3R-mediated Ca2+ responses elicited by 10 microM 5-hydroxytryptamine (serotonin) in canine PASMCs and 100 microM carbachol in human embryonic kidney (HEK)-293 cells were not reduced by 2 microM FLA 365, but they were reduced by 20 microM FLA 365 to 76 +/- 9% of control in canine PASMCs and 52 +/- 1% in HEK-293 cells. Thus, FLA 365 preferentially blocks RyRs with limited inhibition of L-type Ca2+ channels or InsP3R in canine PASMCs.
• Goyal, R., Creel, K. D., Chavis, E., Longo, L. D., & Wilson, S. M. (2006). Maturation of Ca2+ signaling in Ovine pulmonary arterial myocytes. The FASEB Journal, 20(5).
• Ostrovskaya, O., Goyal, R., Mcallister, C. E., Hume, J. R., Pessah, I. N., & Wilson, S. M. (2006). FLA 365 as an inhibitor of ryanodine receptors and L-type Ca2+ channels in arterial myocytes. The FASEB Journal, 20(5).