Valproic acid (2-propylpentanoic acid, VPA) is a clinically used anti-epileptic drug and an effective mood stabilizer. VPA is also a histone deacetylase inhibitor and can induce embryonic malformations in both humans and mice. The mechanism(s) of VPA-induced teratogenicity are not well characterized. The objectives of my study were three fold, to: (i) investigate the effect of VPA on mouse embryonic development, (ii) characterize the putative mechanism(s) of VPA-induced teratogenicity and, (iii) investigate VPA associated epigenetic regulation of Hoxa2 gene in cell lines and in developing embryos. Whole mouse embryo cultures were treated with VPA at doses of 0, 50 (0.35 mM), 100 (0.70 mM), 200 (1.4 mM), and 400 µg/mL (2.8 mM), encompassing the therapeutic range of 0.35 mM to 0.70 mM. Van Maele-Fabry’s morphologic scoring system was used to quantitatively assess embryonic organ differentiation and development. Hoxa2 gene expression was measured by quantitative real-time RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). To assess epigenetic changes on the Hoxa2 gene promoter, DNA methylation was determined by bisulfite (BSP) sequencing and pyrosequencing. Histone “bivalent domains” H3K4me3 (histone 3 lysine 4 trimethylation) and H3K27me3 (histone 3 lysine 27 trimethylation) associated with gene activation repression, respectively, analyzed qChIP-PCR (quantitative chromatin immunoprecipitation-PCR). Telomere length and telomerase activity were analyzed in mouse embryos and in NIH3T3 cell line treated with VPA. Results indicate significantly increased incidence of dysmorphogenesis in embryos (11.8%, 35.3%, 47.0% and 88.3%) exposed to increasing doses of VPA (0.35 mM, 0.70 mM, 1.4 mM and 2.8 mM respectively). Van Maele-Fabry’s quantitative differentiation assessment of developing embryos demonstrated a significantly lower score for the circulation system, central nervous system, craniofacial development and limb development in VPA treated embryos (0.35 mM to 2.8 mM) compared to the untreated control group. Glutathione homeostasis was altered as indicated by decreased total glutathione content and increased GSSG/GSH ratio in all VPA treatment groups. In addition, a dose-dependent inhibition of Hoxa2 gene expression was observed in embryos and in the NIH3T3 cell line exposed to VPA. Pre-treatment with ascorbic acid [1000 µg/mL (5 mM)] restored glutathione level and normalized Hoxa2 gene expression in embryos exposed to VPA. DNA methylation status was characterized on the Hoxa2 gene promoter at the three CpG islands; CpG island 1 (-277 to -620 bp), CpG island 2 (-919 to -1133 bp), and CpG island 3 (-1176 to -1301 bp) in the two cells lines (NIH3T3 and EG7) and in developing embryos. CpG sites remained unmethylated on the Hoxa2 gene promoter in the NIH3T3 cell line which expresses the Hoxa2 gene, whereas these same CpG sites were methylated in EG7 cells that did not express Hoxa2. CpG island 1 is closest to Hoxa2 transcription start site and its methylation status was most affected. In developing embryos, CpG island 1 was found to be highly methylated at E6.5 when Hoxa2 is not expressed, whereas the methylation status of CpG sites on the CpG island 1 declined between E8.5 and E10.5 when Hoxa2 expression is present. VPA induced methylation of several CpG sites on CpG island 1 in NIH3T3 cell line and in E10.5 embryos when Hoxa2 expression was down regulated following VPA exposure. In addition, embryos and the NIH3T3 cell line treated with VPA impacted the “bivalent domains” resulting in increased H3K27me3 enrichment and decreased H3K4me3 enrichment on Hoxa2 promoter. Pre-treatment with ascorbic acid normalized Hoxa2 expression and histone bivalent domain changes and prevented increased DNA methylation following VPA exposure. Moreover, the telomerase activity and telomere length were both impacted by changes in glutathione redox potential induced by VPA. Oxidative stress following VPA treatment reduced telomerase activity and accelerated telomere shortening. These results are the first to demonstrate: (i) a correlation between VPA dose and total morphologic score in the developing mouse embryos. VPA impacted embryonic tissue differentiation and neural system development in the dose range of 0.35 mM to 2.8 mM; (ii) VPA altered glutathione homeostasis in cultured mouse embryos and inhibited Hoxa2 gene expression; (iii) Histone bivalent domains of H3K27 and H3K4 trimethylation and DNA methylation status at the Hoxa2 gene promoter region were altered following treatment with VPA. This appears to be the epigenetic event in transcriptional silencing of Hoxa2 gene expression after VPA exposure; and (iv) Ascorbic acid normalizes glutathione homeostasis, H3K27 and H3K4 trimethylation and DNA methylation status, restoring Hoxa2 gene expression following VPA exposure. Taken together our results show VPA- induced altered glutathione homeostasis, telomere shortening and telomerase dysfunction, and an inhibition of Hoxa2 gene expression leads to developmental abnormalities. Exposure to ascorbic acid had a protective effect on developing embryos exposed to VPA.

No description available.

Coronary heart disease (CHD) remains the single leading cause of natural death worldwide. Despite significant advances in the diagnosis and treatment, CHD accounts for 1 out of every 6 deaths in the United States. Myocardial infarct (MI) as a result of CHD causes irreversible damage to the heart through the loss of viable myocardial tissue. Patients surviving the initial MI are at risk of developing heart failure due to lost contractile function and adverse cardiac remodeling. Improvement in the survival rates for MI have led to an increase in the incidence of heart failure, affecting approximately 5 million people in the United States. Although treatment of heart failure has improved, the mortality rates of heart failure remain high with 1 in 5 dying within the first year of diagnosis and 50% dying within 5 years. The cost of caring for heart failure patients ranks number one in Medicare. Oxidative stress plays an important role in the etiology and pathophysiology of CHD and heart failure. The transcription factor Nrf2 is a master regulator of cellular antioxidant defense mechanisms, controlling the expression of numerous antioxidant and detoxification genes through the Antioxidant Response Element (ARE) in the promoter regions. The cytoprotective effects of Nrf2 have been demonstrated in a variety of organs and disease states; however, the role of Nrf2 in the heart and heart disease has not been defined. The work presented here defines roles of Nrf2 in limiting cardiac injury and the progression to heart failure (Chapter II), protecting cardiac myocytes from oxidative stress through the preservation of mitochondria (Chapter III), and mediating the infarct reducing effects of statins, one of the most prescribed pharmacological agent (Chapter IV). In order to investigate a role of Nrf2 in the pathology of ischemic injury in the heart, a mouse model of ischemia and myocardial infarct by occlusion of the left anterior descending coronary artery was used. Nrf2 knockout mice subjected to ischemia/reperfusion injury experienced a larger infarct size than wild-type mice. Furthermore, mice lacking Nrf2 experienced a higher mortality rate and an accelerated progression to heart failure, indicated by severely compromised contractile function and reduced cardiac output, within 10 days following an MI. Morphological examination revealed maladaptive remodeling, including myocyte hypertrophy, heart enlargement, and dilated left ventricle, in Nrf2 KO mice that was absent in WT mice. Analysis of cardiac function by echocardiogram revealed increased left ventricular mass, increased systolic volume, decreased fraction shortening, reduced ejection fraction, and decreased cardiac output in Nrf2 KO mice. Nrf2 KO mice also demonstrated expression of biomarkers of heart failure, such as expression of fetal gene program, with elevated levels of β-MHC, ANF, and BNP mRNA in the myocardium. Interestingly, a lack of immune cell infiltrate and myofibroblasts as well as a deficiency in collagen deposition were observed in the infarcted region of hearts from Nrf2 KO mice. These data indicate that Nrf2 plays an important role in protecting the myocardium from ischemic injury and the progression to heart failure. Lack of Nrf2 response results in deficiency of wound healing and instead initiation of maladaptive remodeling, leading to heart failure. Mitochondria are key sources of reactive oxygen species (ROS) generation, as well as important targets for ROS-induced cell injury. Cardiac myocytes have the highest content of mitochondria among all cell types and can be particularly susceptible to mitochondrial dysfunction due to the high metabolic demand associated with the contractile function of the heart. With cardiomyocytes (CMCs) isolated from neonatal rats and kept under tissue culture conditions, mitochondria exist in elaborated networks. Such networks were replaced by predominately individual punctate mitochondria 24 hours after exposure to a sublethal dose of H₂O₂. Mitochondrial morphology was altered with membrane swelling and disorganization of inner cristae with areas of condensation. Disrupted mitochondrial morphology was associated with a loss of membrane potential and decreased expression of mitochondrial proteins involved in the electron transport chain, such as cytochrome b and cytochrome c. Nrf2 overexpression prevented H₂O₂ from inducing morphological changes in mitochondria and the reduction of cytochrome b and cytochrome c expresssion. Although Nrf2 is known as a transcription factor regulating antioxidant and detoxification genes, Nrf2 overexpression did not significantly reduce the level of protein oxidation as measured by carbonyl formation. Instead, we found that Nrf2 localizes to the outer mitochondrial membrane, suggesting a direct role of Nrf2 in mitochondrial protection. As further evidence of a direct role in mitochondrial protection, a cell-free system of mitochondria isolated from the myocardium of Nrf2 knockout mice were more sensitive to permeability transition, an indicator of mitochondrial dysfunction. Combined, these data suggest that Nrf2 protects mitochondria from oxidant injury likely through direct interaction with mitochondria. In the clinic, statins are now commonly administered for patients experiencing MI or CHD. Statins have become mainstays in the treatment of hypercholesterolemia and atherosclerosis as inhibitors of the rate limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. In addition, statins have been shown to elicit pleiotropic effects, including plaque stabilization, maintenance of endothelial function, anti-inflammatory actions, and antioxidant capabilities, independent of effects on cholesterol synthesis. Recently, these pleiotropic effects have been implicated in providing acute protection against ischemia and reperfusion injury, which has led to the use of high dose statins clinically before revascularization of an ischemic event. I have found that administration of atorvastatin in mice induced Nrf2 protein levels in the heart, brain, lung, and liver. While atorvastatin reduced infarct size following an MI in wild-type mice, this protective effect was lost in mice lacking Nrf2. Failure of atorvastatin to protect against MI in Nrf2 knockout mice indicates that Nrf2 plays a critical role in mediating the protective effects of acute statin treatment. Nrf2 induction by statins is a novel discovery. In order to understand the mechanism of such statin effect, I used an in vitro cell system, in which a variety of statins, atorvastatin, simvastatin, lovastatin, and pravastatin, were found to elevate Nrf2 protein levels. Elevation of Nrf2 by statins was independent of increased protein stability or transcriptional regulation. Instead, statins increased Nrf2 mRNA association with ribosomal complexes and induced Nrf2 protein through a translational mechanism. Recruitment of Nrf2 mRNA to ribosomes and induction of Nrf2 protein was dependent on activation of PI3 kinase. These studies provide evidence that Nrf2 plays a critical role in protecting cardiac myocytes and the heart from oxidative stress and MI. In the absence of Nrf2, mice experienced worse cardiac injury following MI and quickly advanced to heart failure. Mechanistically, this work has identified a novel role of Nrf2 in preserving mitochondrial morphology and integrity during oxidative stress through a direct interaction with the outer mitochondrial membrane. Finally, a newly defined role of Nrf2 induction by statins in mediating protection against MI by acute statin therapy indicates that modulation of Nrf2 may represent a viable pharmacological target for cardiac protection in humans.

Glucocorticoids are exploited for the treatment of hematological malignancies due to their ability to cause apoptosis in lymphoid cells. Innate and acquired resistance, however, limits their efficacy in the clinic. The mechanisms contributing to resistance are poorly understood. A better understanding of the critical events during glucocorticoid-induced apoptosis are needed in order to develop novel agents that will exploit these critical targets and improve the response to glucocorticoid-based therapies. Previously, using WEHI7.2 murine thymic lymphoma cells, our laboratory demonstrated that the levels of reactive oxygen species (ROS) increase during glucocorticoid-induced apoptosis signaling. WEHI7.2 cell variants with increased catalase exhibit increased resistance to glucocorticoids, suggesting that oxidative stress plays a role in glucocorticoid-induced apoptosis and that increasing the intracellular production of ROS may be a potential strategy for sensitizing lymphoma cells to glucocorticoid treatment. The following studies demonstrate that an increase in H₂O₂ is essential for lymphoma cells to undergo apoptosis and that the ability to remove cellular H₂O₂ protects the cells from glucocorticoid-mediated cell death. The redox-cycling agent, Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin, increased glucocorticoid-induced oxidative stress in WEHI7.2 cells and sensitized the cells to glucocorticoid treatment. MnTE-2-PyP⁵⁺ glutathionylated NF-κB and inhibited its activity. Collectively, these findings suggest that manipulating the redox environment with MnTE-2-PyP⁵⁺ is a promising approach for lymphoma therapy.

Studies conducted for this dissertation utilized a rodent model exposed to single or multiple short duration heat loads in an effort to: 1) elucidate the changes in energy metabolism occurring at the tissue and whole-body level in response to hyperthermia, 2) characterize specific aspects of glucose utilization and hepatic glucose production following a heat load and 3) determine if aspects of mitochondrial function and/or dysfunction might play a role in the metabolic changes that occur in response to heat stress. Study 1 was conducted to determine if rodents exposed to heat stress shared similarities using a bovine heat stress model. Specifically, we were interested in identifying changes in blood metabolites and hormones, as well as gene expression and protein abundance of enzymes associated with energy metabolism in skeletal muscle (type I and type II), liver and adipose tissue. Previous bovine data indicates glucose may be preferentially utilized during heat stress, suggesting alterations in energy metabolism. This study provided evidence that tissue-specific changes occur in response to a heat load and that full glucose oxidation might be reduced, specifically in skeletal muscle where abundance of PDK4 mRNA was increased. Within skeletal muscle, glucose transporters (GLUTs 1 and 4) also tended to be increased in rats exposed to a heat load. Increases in skeletal muscle AMPK-α and PGC-1α as well as increased expression of energy substrate transporters suggests heat stress may impose a cellular energy deficit and/or increased energy demands which subsequently leads to changes in energy metabolism. Few changes were noted in either hepatic or adipose tissue in response to acute heat stress in this pilot study. Study aim of Chapter 3 was to further characterize the effects of heat stress on energy metabolism at the tissue and whole-body level in rats exposed to either 1 or 2 bouts of heat. Rats exposed to a 6 h heat load tended to have higher plasma glucose but reduced insulin levels, compared to thermal neutral controls, suggesting decreased glucose uptake or increased hepatic glucose output. Additionally, although heat stress likely increases whole-body energy demand, plasma NEFA levels were blunted in the early hours following onset of heat, suggesting increased adipocyte insulin sensitivity. Gene expression of enzymes associated with oxidative energy metabolism were increased in the TA (which is comprised primarily of glycolytic muscle fibers) following 2 bouts and in liver following a single bout of heat, while expression of oxidative enzymes were decreased within the soleus (a primarily oxidative muscle type). AMPK mRNA was increased following a single bout of heat in hepatic tissue and after 2 bouts of heat in type I skeletal muscle. AMPK mRNA abundance remained the same following 1 bout but was reduced following 2 bouts of heat within type II skeletal muscle. In the TA, phosphorylated AMPK protein abundance was reduced by HS. Abundance of PGC-1α mRNA was increased in types I and II skeletal muscle but was only numerically increased in liver following heat exposure. These data suggest differences at the transcription level in how heat effects energy metabolism within types I and II skeletal muscle as well as between muscle and hepatic tissue and also suggests a cellular attempt to increase energy production (by all mechanisms) in response to heat exposure. Study 3 (Chapter 4) focused on the effect of a heat load on glucose utilization in skeletal muscle and hepatic glucose production capacity. Similar to study 1, PDK4 expression was increased in types I and II skeletal muscle, while PDK2 expression was increased in hepatic tissue. Within skeletal muscle, increases in PDK expression paralled the increased protein abundance of PDHE1α following heat exposure, implying a decrease in oxidative glucose metabolism. Within the liver, protein abundance of PDH-E1α was reduced following a single heat load, but returned to TN levels after a 2nd heat exposure, suggesting that glucose oxidative metabolism is increased above normal levels after an initial heat exposure, but reduced following multiple heat bouts. Hepatic mRNA abundance for gluconeogenic enzymes were increased, implying an increase in hepatic glucose output capacity. The purpose of Study 4 (Chapter 5) was to determine if heat stress elicits changes on mitochondrial function/dysfunction (i.e. oxidative stress), that may account for changes observed in energy metabolism. Expression of genes associated with antioxidant defense were increased by heat stress, but differed between types I and II skeletal muscle as well as between muscle, hepatic tissue and WBCs. The abundance of mRNA for antioxidant enzymes was increased the greatest, and expression of DNA repair enzymes were also upregulated the most within hepatic tissue due to heat exposure, suggesting either increased damage at the level of hepatocytes or greater defensive capacity following an environmental insult. Taken together, this data provides evidence that heat alters energy metabolism, but these changes are tissue-specific and may be reflective of where damage is occurring, or which tissues are able to adapt and/or compensate for increased energy demands imposed by an environmental insult.

To maintain intracellular redox homeostasis, genes encoding many endogenous antioxidants and phase II detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis- antioxidant responsive elements (AREs) in their promoter regions. Nrf2 has emerged as the pivatol transcription factor responsible for ARE-dependent transcription, and has been shown to play critical roles in hepatotoxicity, chemical carcinogenesis, pulmonary inflammatory diseases, neurodegenerative diseases and aging. Therefore, understanding the molecular mechanism of the Nrf2-dependent cytoprotective system is important for development of drugs for therapeutic intervention.Nrf2 is targeted by Keap1 for ubiquitin-mediated degradation under basal conditions. Upon oxidative stress, distinct cysteine residues of Keap1 are alkylated, leading to inhibition of Keap1 and activation of Nrf2. However, it was not clear how Nrf2 is re-entered into the repression status when redox homeostasis is re-achieved. In this dissertation, we establish that the post-induction repression of Nrf2 is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination/ degradation machinery. We show that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2 signaling; ubiquitination of Nrf2 is carried out in the cytosol; Keap1 nuclear translocation is independent of Nrf2; and the Nrf2-Keap1 complex does not bind the ARE. Collectively, these results suggest that Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE and mediates nuclear export of Nrf2 followed by ubiquitination and degradation of Nrf2 in the cytoplasm.In addition to Keap1-mediated negative regulation, we identified a novel positive regulatory mechanism of Nrf2 mediated by transcription co-activator p300/CBP. We show that p300/CBP directly binds and acetylates Nrf2 in response to oxidative stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner both in vitro and in vivo. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.

Chronic oxidative injury by allylamine induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p21 and p27, and extracellular regulated kinases (ERK1/2) to mediate the proliferative advantage of oxidatively stressed (i.e. allylamine injured) vSMC. Isolated rat aortic SMC from allylamine treated and control rats were cultured on different extracellular matrix (ECM) proteins. Following mitogen restriction, cultures were stimulated with serum with or without inhibitors of NF-kB or MEK. Western blot analysis was performed to identify protein differences between treatment groups. Basal levels of p21 were 1.6 fold higher in randomly cycling allylamine cells than control counterparts seeded on a plastic substrate, a difference lost when cells were seeded on collagen. p27 levels were comparable in both cell types irrespective of substrate. Basal levels of p21 and p27 were 1.4 fold higher in G0 synchronized allylamine cells compared with G0 synchronized control cells seeded on a plastic substrate. Following cell cycle progression, differences in protein levels were not detected. Treatment with 100 nM pyrollidine dithiocarbamate (PDTC) resulted in significant decreases in p21 and p27 in allylamine cells versus control cells following serum stimulation for 9 hours. This decrease was even greater for p21 in allylamine cells when grown on collagen relative to control cells. Alterations in peak and temporal activation of ERK1/2 were observed in allylamine cells seeded on a plastic substrate as compared to control cells, following serum stimulation. Seeding on collagen decreased the enhanced peak phosphorylation of ERK1/2 and increased the sustained activity in allylamine cells compared with control counterparts. Inhibition of ERK1/2 activity resulted in reduced p21 expression in both cells types, but the response was markedly enhanced in allylamine cells, and preferentially observed on a restrictive collagen substrate. We conclude that induction of proliferative (i.e. atherogenic) phenotypes following repeated cycles of oxidative injury involves ERK1/2 activity and modulation of the cyclin dependent kinase inhibitors, p21 and p27, in a matrix-dependent manner.

NF-κB activity is critical in the regulation of atherosclerotic vascular smooth muscle cell (vSMC) phenotypes induced following oxidative injury by allylamine. The present studies were designed to detail dysregulation of NF-κB activity in these altered phenotypes, and to assess the importance of NF-κB in the regulation of osteopontin, a cytokine which modulates atherosclerosis. Increased degradation of IκBα was observed in allylamine-induced atherosclerotic vSMC phenotypes (henceforth referred to as allylamine cells). Enhanced phosphorylation of I-κ-kinases was observed by Western immunoblotting. NF-κB DNA binding activity as assessed by electrophoretic mobility shift assay demonstrated changes in the kinetics and magnitude of induction of binding. Enhancement of NF-κB binding activity was evident in allylamine cells compared to controls when seeded on plastic, fibronectin, and laminin, but not collagen I. Posttranscriptional alterations in Rel protein expression and nuclear localization partly account for changes in NF-κB DNA binding activity. Promoter-specific NF-κB binding profiles suggest altered dimer prevalence as a consequence of the changes in Rel protein expression. The expression of NF-κB regulated genes osteopontin and MMP-2 was enhanced in allylamine-treated aortas, while cyclin D1 and MMP-9 were unchanged. As the importance of osteopontin in atherosclerosis has been described in several models, subsequent studies were designed to assess osteopontin promoter activity. Activity of the osteopontin promoter was significantly reduced in allylamine cells compared to controls as assessed using a luciferase reporter. Deletion analysis suggested the presence of inhibitory cis-acting elements in the regulatory region of the gene. Mutation of these elements, including VDRE, AP-1, NF-κB, and USF1, indicated that NF-κB and USF1 mediate suppression of osteopontin promoter activity in allylamine cells. Decreased serine phosphorylation of immunoprecipitated RelA/p65 was observed in allylamine cells, indicating decreased ability of this protein to transactive gene promoters. NF-κB was found to play a role in suppression of osteopontin promoter activity by collagen I-mediated integrin signaling. These findings suggest that enhancements in NF-κB activity suppress osteopontin promoter activity in oxidant-activated vSMC cultures. Dysregulation of NF-κB activity occurs as a result of altered matrix and intracellular signaling upstream of the nucleus and possibly differential dimer assembly leading to cell-specific profiles of NF-κB-dependent gene regulation.

No description available.

The study objectives were to: (1) assess fetal exposure to oxidative stress by measuring amniotic fluid concentrations of nitric oxide (NO), thiobarbituric acid--reactive substances (TBARS), and ferric reducing antioxidant power (FRAP) and (2) establish whether these concentrations were associated with infant birth weight, gestational age, or oxidative stress-related conditions arising during pregnancy. Frozen amniotic fluid samples were obtained from 654 mothers undergoing amniocentesis for genetic testing during second trimester in Montreal, QC, Canada. Maternal and neonatal characteristics were collected from medical charts and questionnaires and exclusion criteria were applied. ANOVAs and multivariate regression analyses showed that NO, which differed among pre-term, term, and post-term groups, was a positive predictor of gestational age. TBARS were highly correlated with sample storage and were not associated with pregnancy outcome parameters. FRAP positively predicted gender-corrected birth-weight-for-gestational-age. Our study shows that markers of oxidative and nitrative stress in-utero are associated with pregnancy outcomes.