Atherosclerotic Risk of Branched Chain Amino Acids in a Tissue Engineered Blood Vessel Model

点击翻译按钮获取中文摘要

Elevated levels of the branched chain amino acids (BCAA) leucine, isoleucine, and valine are highly correlated with the development of CVD and adverse cardiovascular events, such as heart attacks. Some mechanisms linking BCAA, metabolic disease and heart failure have been documented. However, it remains unclear if BCAAs directly interact with vascular cells to enable CVD onset and progression. Mouse models that have been developed to study CVD and BCAA both have limitations in their applicability to human physiology. Current in vitro models of atherosclerosis are limited to early stages of atherogenesis, and it is challenging to recapitulate more advanced atherosclerotic changes. To address this issue, we will develop a model of an intermediate atherosclerotic lesion using a human tissue engineered blood vessel system (TEBV) model with a disease-pathology collagen extracellular matrix enriched with the glycosaminoglycan chondroitin sulfate (CS), treated with modified low-density lipoprotein (LDL). We will use collagen TEBVs treated with modified LDL as a model for early atherosclerosis. These platforms will be used as a platform to study BCAA mechanism in CVD. The TEBV model is made of collagen or CS-collagen vessels with encapsulated human neonatal dermal fibroblasts serving as the medial cells in the vascular wall and an endothelialized inner lumen of endothelial colony-forming cells (ECFCs). It is perfused with medium at a rate of 2 mL/minute and can be cultured for up to 6 weeks. We have demonstrated that the CS-enriched vessels have an enhanced sensitivity to the inflammatory effects of modified LDL and demonstrate increased vessel vasoactive dysfunction and endothelial-leukocyte interactions compared to collagen TEBVs. We have also demonstrated that elevations of the BCAA within the physiological milieu are sufficient to induce an atherosclerotic phenotype in the endothelium. Preliminary results demonstrate that treatment of ECFCs with elevated BCAA and oxidized low- density lipoprotein causes increased mitochondrial oxidative stress, as well as decreased expression of LC3B, an autophagosome protein. This induced early atherosclerotic events in the TEBV system: impaired endothelium-controlled vasodilation and leukocyte adhesion to the endothelium. Autophagic flux will be further assayed via Western Blot for LC3-I to LC3-II conversion. Vascular cells, especially the endothelium, may be sensitive to the effects of elevated BCAA because they do not significantly contribute to BCAA metabolism, leading to intracellular buildup of metabolites and mitochondrial stress. This will be tested via metabolomic analysis of BCAA and their downstream metabolites in the vascular cells. Using the TEBVs, we will determine the role of BCAA in early versus intermediate atherosclerosis. At the end of the study, we will have an intermediate lesion model of atherosclerosis, the mechanisms linking BCAA to cardiovascular events will become clearer, and potential therapeutic targets for atherosclerosis will be identified.