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Show 143 school of medicine and health sciences UNDERGRADUATE RESEARCH ABSTRACTS Endothelial cells line the vasculature in the circulatory system and are essential to maintain vascular integ-rity. These cells act as a highly selective permeable barrier between the lumen of the cardiovascular system and surrounding tissues, allowing certain materials to pass in and out of the bloodstream. Hyperglycemia compromises the endothelium and induces vascular leakage, which advances diabetic retinopathy and can lead to vision loss if left untreated. The purpose of this experiment is to test whether or not COMP-Ang1, a novel molecule designed to mirror the native Angiopoietin 1 (Ang1) protein, can prevent or rescue the compromised monolayer integrity of human retinal microvascular endothelial cells (HRMVECs). The effects of COMP-Ang1 on HRMVECs were determined using electric cell-substrate impedance sens-ing (ECIS) and Western blots. ECIS works by passing an electric current through the cell monolayer and measuring electrical resistance-the greater the resistance, the greater the integrity of the monolayer. ECIS tested monolayer cell integrity of the HRMVECs before, during, and after treatment with COMP-Ang1, Ang1, PBS, and VEGF. HRMVECs were also treated with COMP-Ang1 and PBS and subsequently harvested for protein content. The protein samples harvested from the cells were analyzed using Western blot assays to identify and quantify VE-cadherin, Src, PhosphoSrc, and GAPDH. PBS was used as a control for all experiments. ECIS results showed that COMP-Ang1 increased barrier resistance when compared to Ang1 and PBS in HRMVECs. VEGF decreased barrier resistance compared to PBS, even in the presence of COMP-Ang1. Western blots showed that COMP-Ang1 decreased Src phosphorylation and increased VE-cadherin when compared to cells treated with PBS. VEGF increased Src phosphorylation and decreased VE-cadherin com-pared to PBS. All Western blot assays showed equal amounts of GAPDH between each sample group, dem-onstrating that protein sample sizes were consistent within sample groups. These results are significant because they correlate increased barrier integrity with decreased Src phosphorylation and increased levels of VE-cadherin stabilization. This is significant because barrier integrity and VE-cadherin are compromised in diabetic retinopathy and COMP-Ang1 could serve as a future therapeutic agent. TESTING THE EFFECTS OF COMP-ANG1 ON ENDOTHELIAL INTEGRITY Kortnie Walker (Balamurali Ambati) Department of Ophthalmology University of Utah Kortnie Walker Balamurali Ambati |
