• Circadian Clock in Angiotensin II Induced Hypertension and Vascular Disease

      Pati, Paramita; Department of Pharmacology and Toxicology (2015)
      Hypertension remains a major risk factor for cardiovascular disease and death. While clinical studies and guideline recommendations underscore the benefits of reducing sodium intake in the treatment of high blood pressure, recent human data suggest that underlying conditions of disease may confound these positive effects of low salt diets. Herein, we examined the influence of circadian dysfunction during experimental hypertension caused by angiotensin II (Ang II), a key peptide in blood pressure regulation. While a low salt diet caused the expected decrease in blood pressure in wild type (WT) mice, mice with disruption of the circadian clock exhibited a paradoxical response to low salt. Mice with disruption in the circadian clock component Period (Period-knockout/KO mice), were abolished in blood pressure rhythm due to an increase in daytime blood pressure. This impairment in blood pressure rhythm in Per-KO mice on the low salt diet was restored to rhythmic oscillation by the angiotensin receptor blocker losartan. Similarly, exogenous administration of Ang Il caused a non-dipping blood pressure phenotype in the Per-KO mice on a normal salt diet, which resulted in pathological thickening of the vasculature indicative of vascular disease. These effects were related to circadian rhythm as impairment in blood pressure caused by low salt was recapitulated in WT mice induced to circadian derangement by a shortened light cycle. Further thickening of the vasculature and increased renin levels were observed in Per-KO mice on a chronic low salt diet but not in WT mice. Moreover, disruption of the Period gene altered ATI receptor expression and other components of the renin-angiotensin system. These data suggest that circadian dysfunction may compromise the benefits of a low salt diet and support recent clinical data that raise caution to sodium restriction as a therapy for hypertension.
    • Deciphering mechanisms of DNA methylation regulation by depletion of the DNA methyltransferases and SETD2

      Tiedemann, Rochelle Lee; Department of Biochemistry and Molecular Biology / Cancer Center (2015)
      DNA methylation (5mC) is a stabile epigenetic mark that confers differential function for gene expression and chromatin accessibility dependent on the context and locality of the mark. Promoter regions populated by CpG islands (CGIs) are highly unmethylated while the remaining ∼80% of CpGs are methylated and distributed across gene bodies, repetitive and transposable elements, and intergenic regions of the genome. The presence and/or absence of particular histone modifications also dictate the patterning of 5mC genome-wide. In cancer, a reversal of 5mC patterns occur in which hypermethylation of tumor suppressor gene CGIs confers gene silencing, and hypomethylation of repetitive and transposable elements contribute to genomic instability. The mechanisms by which 5mC becomes aberrantly regulated in cancer remain unknown. In this study, direct and indirect mechanisms of 5mC regulation were investigated. To understand the direct regulation of 5mC genome-wide, we depleted cell line models of the DNA methyltransferases (DNMTs) that are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) 5mC patterns. Profiling of 5mC patterns on the Illumina HumanMethylation450 BeadChip revealed a unique antithetical relationship between DNMT1 and DNMT3B for the regulation of both 5mC and DNA hydroxymethylation (5hmC) across gene bodies. DNMT3B mediated nonCpG methylation, while DNMT3L influenced the activity of DNMT3B toward nonCG versus CpG site methylation. DNMT3B depletion induced 5mC patterns that closely resemble those observed during cellular differentiation and occurred across gene bodies of highly expressed, H3K36me3-marked genes. SETD2, the histone methyltransferase responsible for H3K36me3 establishment across active gene bodies, was determined to influence the guidance of DNA methylation genome-wide through an indirect mechanism. SETD2 knockout induced widespread loss of H3K36me3 that did not coincide with changes in 5mC. However, paradoxical gains in H3K36me3 significantly induced hypermethylation and upregulation of underlying genes. Genes marked exclusively by the poised enhancer mark, H3K4me1, were commonly targeted for this epigenetic phenotype. DNA methylome profiling of loss-of-function SETD2 mutated clear cell renal cell carcinoma, papillary renal cell carcinoma, and lung adenocarcinoma tumors confirmed the predominance of the hypermethylation phenotype upon loss of SETD2. Collectively, these studies provide novel insight to understanding the regulatory mechanisms by which 5mC patterns are conferred.
    • Early adolescents' physical activity and nutrition beliefs and behaviors in an urban cluster in the southeastern United States

      Hawks, Miranda R.; Department of Physiological and Technological Nursing (2016)
      Obesity in early adolescents is a significant public health problem that has adverse health consequences, to include increasing the risk of developing type two diabetes and hypertension. Factors such as the environment, nutrition, and physical activity contribute to obesity in early adolescents. The purpose of this ethnographic study was to explore the physical activity and nutrition beliefs and behaviors of early adolescents in an urban cluster in the southeastern part of the United States. The researcher recruited early adolescents at a community organization and collected data using three ethnographic methods: semi-structured interviewing, participant observation, and collection of artifacts. Data were analyzed using constant comparative analysis to shed light on the meaning of early adolescents’ communications about their physical activity and nutrition beliefs and behaviors. Themes that emerged from data analysis included recognizing benefits of physical activity and healthy eating, family influences, connecting with the community, peer influences, electronic media influences, and developing a sense of self. This study contributes to nursing science in three ways. First, all early adolescents recognized both physical activity and healthy eating as beneficial for promoting their health and improving the quality of their lives. Second, early adolescents described their mothers as the most influential family member for both their physical activity and healthy eating behaviors. Third, the community organization was identified as the main facilitator of early adolescents’ physical activities within their immediate environment outside their home. These findings explain three different points of entry that the nursing community can use, separately or together, for their health promotion strategies to encourage physical activity and healthy eating among early adolescents.
    • Involvement of arginase upregulation in diabetes- and angiotensin II-induced vascular dysfunction

      Bhatta, Anil; Department of Pharmacology and Toxicology (2015)
      Cardiovascular disease (CVD) is the number 1 killer of men and women in the United States and the world. Diabetes, hypertension, obesity, and aging are some of the risk factors for CVD. A major cause of morbidity and mortality in CVD is vascular dysfunction, which progresses rapidly as the risk factors progress. Vascular dysfunction is characterized by a constellation of blood flow reducing pathologies, including impaired vasorelaxation and elevated arterial stiffening. The mechanisms leading to these vascular abnormalities are not well understood. We tested the hypothesis that arginase, an enzyme in the urea cycle, mediates vascular dysfunction in hypertension and obesity related diabetes. Arginase (ARG) can compete with nitric oxide (NO) synthase for their common substrate, L-arginine. Increased arginase can also provide more ornithine for synthesis of polyamines via ornithine decarboxylase (ODC) and proline/collagen via ornithine aminotransferase (OAT), leading to vascular cell proliferation and collagen formation, respectively. We hypothesized that elevated arginase activity is involved in Ang II-induced vascular dysfunction and that limiting its activity can prevent these changes. We tested this by studies in C57BL/6J mice lacking one copy of the ARG1 gene that were treated with Ang II (1 mg/kg/day, 4 weeks). We demonstrated that Ang II induces smooth muscle cell proliferation, collagen synthesis, and arterial fibrosis and stiffness via a mechanism involving increased arginase activity. Furthermore, we examined the role of arginase in vascular dysfunctions and pathologies associated with obesity-related type 2 diabetes in mice fed with high-fat/high-sucrose (HFHS) diet for 6 months. This model produced a clinical presentation and pathophysiological relevance to the human condition in obesity related type 2 diabetes. We demonstrated that HFHS diet impaired endothelial dependent vasorelaxation and increased arterial stiffness in WT mice, but not in mice treated with arginase inhibitor ABH. Endothelial cell specific knockout of ARG1 (EC-A1-/-) in mice also prevented HFHS induced vascular dysfunctions. Aortic perivascular collagen deposition was significantly higher in HFHS mice compared to normal diet. Furthermore, marked increase in vascular cell adhesion molecule expression and macrophage infiltration into the aortic walls was observed with HFHS diet. Additionally, plasma lipid peroxidase activity, a measure of systemic oxidative stress, was also markedly increased in HFHS mice. These changes were prevented in ABH treated mice and EC-A1-/- mice. These studies suggest that enhanced ARG1 activity promotes vascular dysfunctions associated with elevated Ang II levels or obesity related diabetes.
    • Role of DNA methyltransferases in maintaining mammary stem/progenitor and cancer stem cells

      Pathania, Rajneesh; Department of Biochemistry and Molecular Biology (2015)
      Breast cancer is the leading cause of cancer death in women worldwide and it affects one in eight women in western countries. Like other human cancers, breast cancer also consists of cellular hierarchy, and heterogeneous. However, the cancer cell of origin and how a normal self-renewal pathway turns into abnormal self-renewal signaling are not known. DNA methylation provides a potential epigenetic mechanism for the cellular memory and heterogeneity, which needed to preserve the tumorigenic potential through repeated cell divisions. Further DNA methylation plays an essential role in stem/progenitor cell maintenance and provides a potential epigenetic mechanism for maintaining cellular memory and heterogeneity during self-renewal. However, the specific role of DNMTs in maintaining mammary stem cells (MaSC) and cancer stem cell (CSC) in a constantly replenishing organ, like mammary glands, is not yet known. Here, we show that Dnmt1 is essential for mammary gland development and indispensable for terminal end bud development and that mammary-gland specific Dnmt1 deletion in mice leads to significant reduction in mammary stem/progenitor cell formation. Moreover, Dnmt1 deletion almost completely abolishes Neu-Tg- and C3(1)-SV40-Tg- driven mammary tumor formation. The reduced tumor incidence observed in Dnmt1 deleted mouse is associated to significant reduction in cancer stem cell formation. These observations were recapitulated using pharmacological inhibitors of DNMTs in Neu-Tg mice in vivo. Further, we show that there is a substantial increase in DNMT1 expression when mammary stem/progenitor cells turn into tumor initiating cells. Using genome-scale methylation approach, we found that hypermethylation of genes involved in development and cell commitment pathways impart immortality and autonomous growth to the cancer stem cells. Moreover, our study provides evidence that stem cells, in addition to luminal progenitor cells, are susceptible for genetic and epigenetic modification and associated with chemotherapeutic resistance. Thus, combination of DNMT and HDAC inhibitors can be used as a therapeutic strategy to block mammary tumor formation and to overcome drug resistance by inhibiting CSCs. These findings improve our understanding of abnormal self-renewal associated with cell of origin, and highlight novel methylation markers that have the potential to serve as useful diagnostic tools and therapeutic targets in early detection of breast cancer.
    • RPE as a key modulator of oxidative stress, inflammation, and hemoglobin production in retinal health and disease

      Promsote, Wanwisa; Department of Biochemistry and Molecular Biology (2015)
      The retinal pigment epithelium (RPE) is a highly specialized monolayer of pigmented cells located between the neural retina and the choroid in the eye. RPE provides structural support and functions that are crucial for maintaining healthy retina and its visual activities. Damage to or dysfunction of RPE is implicated in the pathogenesis and progression of several ocular diseases. This study focuses specifically on the role of RPE in the pathophysiology of two ocular diseases: age-related macular degeneration (AMD) and sickle cell retinopathy (SR). This dissertation first gives an overview of the eye with a detailed description of the structure and function of the neural retina and RPE, followed by literature reviews on the pathophysiology o f and current therapeutic options for AMD and SR. The rationale and significance of the study with respect to each disease is discussed separately. Then, the manuscripts detailing the major research findings are presented. Last, the discussion and summary of the overall research findings and the conclusion of the study are provided.