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

Abstract

The purpose of this work is to determine if branched chain amino acids (BCAA) could have a causative role in the development of atherosclerosis. Atherosclerotic lesions occur in the vasculature and mediate the progression of cardiovascular disease (CVD). Advanced atherosclerotic lesions can lead to heart attacks or strokes. Conflicting evidence from previous studies has made it difficult to understand if BCAA help or hurt cardiovascular health, and it is not known if other pro-atherosclerotic factors cooperate with BCAA to accelerate atherosclerosis. While studies in human patients have shown that there is a correlation between BCAA levels in the blood and the development of diseases like metabolic syndrome and CVD, we cannot conclude that BCAA cause these diseases from association studies alone. Additionally, some studies in animals have shown that supplementation with BCAA supports cardiovascular health. Therefore, we need to determine if there is a mechanistic link between BCAA levels and atherosclerotic disease processes in human cells. This will also help us determine if BCAA could be a mechanistic link between metabolic syndrome and CVD.In this work, we use a tissue engineered blood vessel (TEBV) model to determine the role of BCAA in the development of atherosclerosis. The TEBV model is an artificial blood vessel made of a collagen-based scaffold and populated with vascular cells, using similar tissue architecture and environmental stimuli of an artery in the human body. The TEBV system models the processes that occur in atherosclerosis, such as inflammation, loss of vasomotor tone, and interactions between white blood cells and vascular cells. Measuring these processes then allows us to predict what effect a novel risk factor, such as BCAA, would have on vascular health in the human body.In chapter 2, we develop an “intermediate stage” lesion model in the TEBV system. Atherosclerotic lesions develop over many years, and since metabolic syndrome is often diagnosed in adults, many patients will have existing lesions. Therefore, it is important to expand upon existing models of early atherogenesis to include features that occur in later disease stages, such as remodeling of the vessel wall. We recapitulate the increase of a carbohydrate-based molecule called chondroitin sulfate (CS) that occurs in the atherosclerotic extracellular matrix in our TEBV model. While many studies have shown that CS enhances the development of atherosclerosis by affecting processes like lipid retention in the vessel wall and inflammation, there have not been many in vitro disease models that include the effects of CS-remodeling that occurs in the body In our work, we demonstrated that enriching the TEBV extracellular matrix with pathological levels of CS leads to an enhanced atherosclerotic response to treatment with modified low-density lipoprotein (LDL), including increased VCAM expression, a marker of inflammation in endothelial cells, and increased white blood cell adhesion to the vessel wall.In chapter 3, we tested the effects of BCAA treatment on endothelial cell and TEBV health. We cultured cells and TEBVs in a low-BCAA medium that reflected the BCAA levels that occur in human serum. While a few other studies have looked at the effects of BCAA on human vascular cells, they did not use physiologically relevant levels of BCAA in their untreated controls, and used much higher levels of BCAA doses to test their effects. In our studies, we found that BCAA affect several key processes related to endothelial health, inducing oxidative stress in the mitochondria, inducing increased expression of redox-balancing enzymes, and slowing autophagy. This was consistent with results in TEBV experiments, where we saw that BCAA cooperate with another pro-atherosclerotic agent, oxidized LDL, to induce vasodilation dysfunction and increased white blood cell adhesion to the vessel wall.In chapter 4, we evaluated the hypothesis that slowing BCAA catabolism is sufficient to induce buildup of BCAA in vascular cells, leading to an atherosclerotic phenotype. To slow BCAA catabolism, we used a dCas9-KRAB construct to repress the gene PPM1K. PPM1K plays a critical regulatory role in modulating BCAA levels in the cell by activating the rate-limiting enzyme in the BCAA metabolic pathway and stimulating BCAA breakdown. We found that repressing PPM1K effectively alters the active state of its target enzyme, BCKDH, and increases glutamine and serine levels in iPSC-derived endothelial cells. In TEBVs, we found that PPM1K repressed-endothelium induces a differential response to oxLDL treatment in causing vasodilation dysfunction, compared to the vehicle control. Thus, there may be a role for BCAA metabolism in enhancing an atherosclerotic phenotype induced by other pro-atherosclerotic factors.In summary, we determined that BCAA can contribute to an atherosclerotic phenotype, specifically by affecting endothelial cell health. This conclusion is supported by our observations that BCAA affect several key molecular and functional markers of endothelial health, including mitochondrial oxidative stress, autophagy, vasodilation function, and white blood cell adhesion to the endothelium. Importantly, in TEBVs, the presence of other pro-inflammatory factors, such as oxLDL, enhanced these effects. Future research should aim to identify which of these processes may be a suitable target to interrupt the atherosclerotic risk of BCAA.