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

支链氨基酸(BCAA)亮氨酸、异亮氨酸和缬氨酸水平的升高高度相关 随着心血管疾病的发展和不良心血管事件，如心脏病发作。一些机制 支链氨基酸、代谢性疾病和心力衰竭之间的联系已经被记录在案。然而，目前还不清楚是否 支链氨基酸直接与血管细胞相互作用，使CVD的发生和发展成为可能。具有以下特性的鼠标模型 用于研究心血管疾病和支链氨基酸，但它们在人体生理学方面的适用性都有局限性。 目前动脉粥样硬化的体外模型仅限于动脉粥样硬化形成的早期阶段，这是具有挑战性的 重述更高级的动脉粥样硬化改变。为了解决这个问题，我们将开发一个模型 使用人组织工程血管系统(TEBV)模型的中度动脉粥样硬化病变 疾病病理胶原细胞外基质富含糖胺聚糖软骨素硫酸盐(CS)， 用改良低密度脂蛋白(LDL)治疗。我们将使用经修饰的低密度脂蛋白处理的胶原蛋白TEBV作为 早期动脉粥样硬化模型。这些平台将作为研究CVD中支链氨基酸机制的平台。 TEBV模型是由胶原或CS-胶原血管与包裹的人类新生儿真皮制成的 成纤维细胞作为血管壁的中层细胞和内皮细胞的内腔 集落形成细胞(ECFC)。它以2毫升/分钟的速度灌流，可培养长达 6周。我们已经证明，富含CS的血管对 修饰的低密度脂蛋白的炎症作用和血管活性功能障碍的增加 与胶原蛋白TEBV相比，内皮细胞与白细胞的相互作用。我们还证明了 生理环境中的支链氨基酸足以诱导动脉粥样硬化表型。 内皮细胞。初步结果表明，高支链氨基酸和氧化低碳源对ECFC的处理效果显著。 密度脂蛋白导致线粒体氧化应激增加，以及LC3B表达减少， 一种自噬蛋白。这导致了TEBV系统的早期动脉粥样硬化事件：受损 内皮控制的血管扩张和白细胞与内皮的黏附。自噬通量将进一步 通过Western Blot检测Lc3-I向Lc3-II的转化。血管细胞，特别是内皮细胞，可能是 对支链氨基酸升高的影响很敏感，因为它们对支链氨基酸的新陈代谢没有显著贡献， 导致代谢物在细胞内积聚和线粒体应激。这将通过代谢组学进行测试 血管细胞中支链氨基酸及其下游代谢物的分析。使用TEBV，我们将确定 支链氨基酸在早期和中期动脉粥样硬化中的作用。在研究结束时，我们将有一个 动脉粥样硬化的中间病变模型，将支链氨基酸与心血管事件联系起来的机制将 变得更加清晰，动脉粥样硬化的潜在治疗靶点将被确定。

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.