Abstract 3131: An Investigation Into The Interplay Between Epha2 And Inflammatory Responses In Vascular Smooth Muscle Cells

Abstract

Cardiovascular disease continues to be a leading cause of death in the United States. A major underlying cause of cardiovascular events such as stroke and myocardial infarction is atherosclerosis. Notable mediators in the pathogenesis of atherosclerosis include vascular smooth muscle cells (VSMCs) and inflammation. During atherogenesis, the SMCs undergo phenotypic change from a quiescent state to a synthetic, proliferative state. The synthetic SMCs drive neointima formation which influences the stenosis characteristic of atherosclerosis. Signaling of a receptor tyrosine kinase called EphA2 has been previously determined to have a role in SMC phenotypic change. It was found that a global and SMC specific knockdown (KD) of EphA2 resulted in decreased plaque formation, suggesting the role of EphA2 expression in promoting SMC proliferation. Inflammation is another important factor that contributes to plaque formation. Oxidized low-density lipoproteins (LDLs) have been found to also cause VSMCs to undergo change to a synthetic phenotype, one that resembles macrophages. Cytokines released by macrophages orchestrate the pro-inflammatory response that affects the development and stability of atherosclerotic plaques. Considering the important role inflammation also serves in the progression of plaque formation, we investigated the relationship between EphA2 and VSMC-mediated inflammatory response. Cre-flox breeding was performed to achieve EphA2 knockout (KO) specifically in VSMCs of 8-10-wk old ApoE -/- mice. Successful KO was confirmed with PCR and Western blot. Si-RNA was transfected to achieve KD in human SMCs. The cells were then treated with cytokines, including IL-1 beta, TNF-alpha, IL-6, IL-33, and interferon-gamma, followed by RT-qPCR analysis to measure expression of inflammatory genes. To assess cytokine secretion, we utilized a cytokine array to measure presence of the cytokines of interest. Protein analysis to quantify expression of inflammatory proteins in KO vs control cells was done via Western blot. We observed that the loss of EphA2 causes significant reductions in the production of several proinflammatory cytokines and chemokines, indicating its role in mediating VSMC inflammatory responses.