Inflammation contributes to cardiovascular complications arising from atherosclerosis and is a validated clinical target, but the underlying mechanisms regulating chronic T cell interactions with the arterial wall remain poorly understood. Our lab has identified thousands of endothelial alternative splicing events regulated by low and disturbed blood flow, many of which effect genes involved in adaptive immune responses. In silico and in vitro analyses identified Elavl1, an RNA-binding protein implicated through as yet unknown mechanisms in human atherosclerosis, as the top candidate splice factor regulating these changes. Preliminary data using a novel endothelial cell-specific Elavl1 knockout (EC-KO) reveal that Elavl1 regulates hundreds of alternative splicing events in the inflamed arterial intima of plaque. These events were enriched in genes associated with adaptive immune responses and were associated with reductions in plaque development and T cell numbers in two models of atherogenesis. We observed no T cell defects peripherally in blood or lymph node, leading us to hypothesize that that endothelial Elavl1 may be an important regulator of local T cell interactions with atherosclerotic intima. As the primary personnel on this project, I will be investigating this hypothesis with the following aims. First, we will extend our preliminary data to characterize the role of endothelial Elavl1 in atherosclerotic plaque development, and T cell recruitment, retention and phenotype. We will model atherosclerosis by inducing chronic hyperlipidemia. After three months we will adoptively transfer and systemically activate T cells and collect aortas early to assess recruitment and later to assess retention. As sex is an important variable, our cohorts will be powered to detect significant differences in both males and females. Second, we will determine whether Elavl1 regulates endothelial activation and T cell interactions through nuclear (splicing) or cytoplasmic (stabilization) functions. We will generate an immortalized mouse Elavl1 EC-KO line and then clone rescue constructs localized to the nucleus or cytoplasm to assess effects of Elavl1 splicing or stabilization on inflammatory signaling. We will leverage the inflammatory NFKB pathway, using flow cytometry to read changes in induction of ICAM and VCAM as a result of nuclear/cytoplasmic localization. We will also use flow chambers to pass T cells over ECs to determine Elavl1 effects on T cell attachment.
|Program type||Predoctoral Fellowship|
|Effective start/end date||01/01/2020 → 12/31/2021|