Date of Graduation

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Kavdia, Mahendra

Committee Member

Beitle, Robert R. Jr.

Second Committee Member

Kluess, Heidi A.

Third Committee Member

Erf, Gisela F.

Keywords

Biological sciences; Health and environmental sciences; Endothelial cells; Hyperglycemic cells; Nitric oxide; Superoxide pathways

Abstract

Cardiovascular complications arising from diabetic hyperglycemia represents one of the leading causes of death and greatest public health challenges of modern societies. Despite state-of-the-art glucose control, diabetic patients remain at a markedly increased risk of cardiovascular disease. The loss of endothelial function (the development of diabetic endothelial dysfunction) has been implicated in the development of numerous diabetic cardiovascular diseases. The endothelial cell produces many vasoactive substances, hormones and cytoprotective biological factors. Endothelial cells are also involved in and affected by the initiation of inflammatory responses through the release and interaction of cytokines and other immune system molecules. Therefore, regulation of these signaling molecules is extremely important to the health of the vascular endothelium and, consequently, damage to the cells ability to control vessel tone and inflammation is a known hallmark to numerous cardiovascular diseases. Much of recent research attention is directed towards the loss of the ability of the diabetic vasculature to produce nitric oxide (vasodilator and anti-inflammatory hormone, a key component of vascular homeostasis). The observation that endothelial cells in diabetes fail to produce sufficient amount of nitric oxide and fail to relax in response to the endothelium-dependent vasorelaxants (e.g. acetylcholine, bradykinin, shear stress, etc.) has been documented by multiple studies, both in animal models of the disease and in human studies. In this dissertation, we investigated the molecular and enzymatic mechanisms associated with the loss of nitric oxide bioavailability and increase in oxidant formation using a hyperglycemic human umbilical vein endothelial cell model. Our results indicate that while hyperglycemia decreases overall nitric oxide levels, generation of nitric oxide is paradoxically increased, validating previous modeling data published by our lab. Furthermore, we were able to indirectly confirm this concomitant increase in superoxide and nitric oxide by showing a significant increase in the formation of nitrotyrosine in high glucose exposed endothelial cells. This illustrates that the parallel increase in superoxide and nitric oxide lead to increased reaction with one another, resulting in higher levels of the cytotoxic peroxynitrite molecule. To better understand the effects of angiotensin II and high glucose on gene regulation of oxidant generating enzymes involved in oxidative and nitrosative stress pathways, we performed real-time quantitative PCR for NADPH oxidase subunits and nitric oxide synthase isoforms in HUVEC's. Results from our studies show that stimulating effects of angiotensin II on the activity of endothelial cell NADPH oxidases is enhanced in high-glucose exposed HUVEC's. We also show that hyperglycemic endothelial cells are more sensitive to Ang II interaction, resulting in lower levels of nitric oxide bioavailability and increased nitrotyrosine formation. Our results also provide insight into the gene regulation of NADPH oxidase, eNOS and iNOS. Data shows that angiotensin II increases NADPH oxidase and iNOS mRNA levels in high-glucose exposed HUVECs, while eNOS expression is relatively unchanged. This further validates the hypothesis that high glucose initiates a protective response in endothelial cells by upregulating nitric oxide producing enzymes, iNOS, in an attempt to counteract/scavenge the increased production of superoxide by NADPH oxidase. This protective measure only exacerbates the oxidative and nitrosative stress environment of the cell, leading to increased cell damage and/or apoptosis. Studies in this dissertation will help clarify the molecular mechanisms and interactions involved in hyperglycemia induced oxidative and nitrosative stress, providing improved focus for treatment design towards improving/reversing high glucose induced endothelial cell dysfunction.

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