Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, which predisposes to ischemic cardiovascular events. These vascular disturbances may increase morbidity and mortality in diabetic patients. We have shown that down-regulation of small (SKCa) and intermediate (IKCa) conductance calcium-activated potassium channels contributes to diabetic-induced endothelial dysfunction in human coronary microvasculature. Recent evidence suggests that Nox and dysfunctional mitochondria mutually stimulate to enhance mitochondrial ROS (mROS) production and PKC expression/activation and play a pivotal role in endothelial dysfunction during diabetes. However, the precise mechanisms responsible for metabolic down- regulation of SKCa/IKCa and coronary endothelial dysfunction are still undefined. There has been limited investigation into NADH-Nox, mROS, and PKC signaling in diabetic regulation of endothelial SKCa/IKCa at molecular and cellular levels. Interestingly, our group demonstrated that elevation in intracellula NADH results in a significant decrease in SKCa/IKCa, and the lack of changes in SKCa/IKCa gene/protein abundances in the setting of diabetes suggests that the effect is post-transcriptional. The goal of this project is to investigate how persistent oxidative stress and PKC over-expression/activation during diabetes negatively regulates SKCa/IKCa channels of human endothelial cells and endothelial function in the human coronary microvasculature. We hypothesize that persistent overproduction of reactive oxygen species (ROS) via NADPH oxidase (Nox) and dysfunctional mitochondria and PKC in the setting of diabetes will result in 1) down-regulation of endothelial SKCa/IKCa, 2) impairment of endothelial function; and that 3) inhibition of Nox and mROS and PKC may potentiate SKCa/IKCa activator-induced endothelial protection of the human coronary arterioles against a simulated cardioplegia ischemia/reperfusion (I/R) injury. Using human atrial tissue samples, isolated human coronary arterioles, and coronary endothelial cells, we will test our hypothesis by completing 4 specific aims.
Aim 1 : To investigate the molecular mechanisms by which persistent over-expression/activation of Nox and NADH during diabetes results in mitochondrial dysfunction and PKC activation, leading to SKCa/IKCa downregulation and coronary endothelial dysfunction.
Aim 2 : To elucidate the molecular mechanisms by which persistent increases in mROS from the mitochondrial complex I, II, III and IV are required for diabetic down- regulating SKCa/IKCa, and endothelial function.
Aim 3 : To define the signaling pathways by which persistent PKC activation during diabetes negatively modifies SKCa/IKCa, and endothelial function.
Aim 4 : To study if selective inhibitors of Nox or mROS or PKC may potentiate SKCa/IKCa activator-induced endothelial protection of the coronary arterioles against a simulated cardioplegia I/R injury. The present study will hopefully lead to novel drug therapies to preserve coronary endothelial function for diabetic or non-diabetic patients with coronary artery disease during cardiac surgery.

Public Health Relevance

The goal of the proposed research is to determine that persistent overproduction of NADPH oxidase (Nox), mitochondrial reactive oxygen species (mROS) and over-expression/activation of PKC during diabetes mellitus result in down-regulation of the small (SKCa) and intermediate (IKCa) conductance of calcium-activated- potassium channels and human coronary endothelial function and to examine the potentially protective effects of exogenous KCa activators, and/or the inhibitors of Nox, mROS and PKC on the coronary arteriolar function against ischemia and reperfusion injury. The present study will hopefully lead to novel drug therapies for protecting human coronary microvasculature during cardiac surgery or in ischemic heart disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
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Fleg, Jerome L
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Rhode Island Hospital
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