Presenter: Revi Brown, College of Health, Nutrition and Integrative Physiology
Authors: Revi Brown, Seul-Ki Park, Rajeshway Ghosh, Parhum Namdaran, Anwar Tandar, Sihem Boudina, J David Symons
Faculty Advisors: John David Symons, College of Health, Nutrition and Integrative Physiology
Institution: University of Utah
Autophagy is a conserved intracellular degradation / recycling pathway designed to clear damaged cytosolic proteins. My laboratory showed earlier that endothelial cell (EC) autophagy is repressed in primary arterial ECs from preclinical and human models of healthy aging that concurrently display impaired activating phosphorylation of endothelial nitric oxide (NO) synthase at serine 1177 (p-eNOSS1177) and NO generation. The purpose of my project is to test the hypothesis that autophagic flux and function are impaired in arterial ECs from patients with cardiovascular disease. To do so, the sheath that is otherwise discarded at the completion of an arterial catheterization is obtained, and the adherent cells are collected and plated on 8-well chamber slides. For each patient, 4-wells are treated for 60-min with bafilomycin (BafA1; to estimate autophagic flux) or dimethylsulfoxide (DMSO, the vehicle for bafilomycin; to estimate steady state autophagy). Two of 4-wells from the BafA1 and DMSO treatments are concurrently incubated with diaminofluorescein (DAF) to estimate NO generation, followed by staining with immunofluorescent antibodies for VE-cadherin (to identify ECs) and DAPI (to identify nuclei). NO generation is quantified by immunofluorescence in at least 20 cells that are VE-cadherin and DAPI positive using confocal microscopy. The remaining 2-wells from each treatment are fixed with paraformaldehyde and stored at -80°C. At the time of analysis, samples are thawed and treated with immunofluorescent antibodies for VE-cadherin, DAPI, p-eNOSS1177, and LC3B and p62 (to estimate autophagy). Indexes of autophagy and eNOS activation are quantified in at least 20 cells that are VE-cadherin and DAPI positive using confocal microscopy. After obtaining results from 100 patients, statistical analyses will be completed to test the hypotheses that autophagic flux and NO generation are repressed in : (i) diabetic vs. non-diabetic patients, (ii) hypertensive vs. non-hypertensive patients; and (iii) older vs. younger patients.