Endothelial Cell Dysfunction in Oxidative Stress Models

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DESCRIPTION (Provided by applicant): Endothelial cell dysfunction is a primary basis of cardiovascular disease including diabetes mellitus. Evidence suggests that supplemental L-arginine (L-arg) is therapeutically useful in reversing endothelial dysfunction and treating cardiovascular disease, but the mechanism of this effect is unknown. Therefore, we are studying the impact of oxidative injury on endothelial cell transport of L-arg and how it relates to endothelial dysfunction by using experimental models of diabetic coronary artery disease. The normal function of the vascular system depends critically on nitric oxide (NO) production by vascular endothelial cells (EC). However, in conditions associated with oxidative vascular injury such as diabetes mellitus, atherosclerosis, and hyperhomocystememia, excess formation of reactive oxygen species can lead to endothelial dysfunction and reduction in NO bioavailability. NO is produced by NO synthase (NOS) from its substrate L-arg. When L-arg availability to NOS is limiting, NOS acts principally upon 0, to form superoxide (O-), which rapidly combines with NO to form peroxynitrite (ONOO). ONOO- and 02+formation can lead to further formation of O NOS due to oxidation of BH4 (tetrahydrobiopterin), a critical co-factor for NOS. In EC, supply of L-arg to NOS depends mainly on the function of a specific transporter, system y+. Our data show that continued NO oxidant exposure inhibits system y transport of L-arg, reducing availability of L-arg and leading to formation of O2 This EC pathology is reversed with supplemental L-arg. We hypothesize that endothelial cell injury mediated by reactive oxygen species (ROS) reduces L-arg transport function. This reduces L-arg uptake and shifts NOS activity from NO production to O2 - production, leading to further compromise of the L-arg transporter. These deleterious effects can be prevented with supplemental L-arg. Our specific aims will test these hypotheses and further characterize the regulation of L-arg transporter. Aim 1. HYPOTHESIS: Chronic exposure 10 ROS causes dysfunction of the L-arg transporter. To test this hypothesis, we will determine the effects of chronic exposure to NOS agonists, NO donors, 02+-, ONOO- on uptake of [3H]L-arg in A) human coronary artery ECs and B) isolated rabbit hearts perfused by the Langendorff procedure. Aim 2. HYPOTHESIS.- Reduction of L-arg uptake shifts NOS activity from NO production to O - production leading to further compromise of the L-arg transporter. We will use the oxidant treatment protocols of aim 1 to correlate basal and NOS agonist-stimulated EC production of NO, O2 - and ONOO- with L-arg transport activity. Aim 3. HYPOTHESIS: Oxidant exposure alters transporter protein expression subcellular distribution and/or molecular interactions with eNOS. Recent studies indicate the principal supply of L-arg to eNOS occurs within caveolae where the L-arg transporter protein CAT1 interacts with eNOS. Thus, oxidant exposure may inhibit L-arg transport by altering CAT! expression levels, subcellular compartmentalization and/or protein-protein interactions with eNOS. Interactions of these systems will be tested by experiments exposing HCAEC to the above oxidant treatments and determing the effects on CAT expression, subcellular distribution and molecular interactions with eNOS by using immunoprecipitation, immunoblotting, subcellular fractionation and confocal microscopy. Aim 4. HYPOTHESIS: High glucose/ diabetes causes endothelial dysfunction and reduces the bioavailability of NO by increasing formation of O2 - and ONOO which alters function of the L-arg transporter, oxidizes tetrahydrobiopterin, and shifts eNOS activity from NO to 02 - production. This hypothesis will be tested by the following experiments: A) determining the effects of high glucose/diabetes on L-arg transport in relation to eNOS expression and activity and formation of NO of 02 - and ONOO- in HCAEs exposed to high glucose or control conditions and in the coronary circulation isolated from diabetic rabbit hearts; and B) determining whether supplemental L-arg is effective in preventing the effects of high glucose/diabetes on the above parameters.