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

组织工程血管模型中支链氨基酸的动脉粥样硬化风险

基本信息

批准号：
10753482
负责人：
Ellery Jensen Jones
金额：
$ 0.89万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2023-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10753482
关键词：
Acceleration Address Adhesions Affect Amino Acids Arterial Fatty Streak Atherosclerosis Autophagocytosis Autophagosome Biological Biological Assay Biological Models Blood Blood Vessels Branched-Chain Amino Acids Cardiovascular Diseases Cardiovascular system Cause of Death Cells Chondroitin Sulfates Clinical Collaborations Collagen Dermal Development Diabetes Mellitus Disease Disease Progression Disease model Encapsulated Endothelium Environment Event Extracellular Matrix Fibroblasts Foam Cells Functional disorder Glucose Goals Health Heart Diseases Heart failure Human Hydrogels Impairment Incidence Inflammation Inflammatory Insulin Resistance Isoleucine Lesion Leucine Leukocytes Link Lipids Lipoprotein Binding Low-Density Lipoproteins Medial Mediating Metabolic Metabolic Diseases Metabolic syndrome Minority Groups Mitochondria Modeling Molecular Myocardial Infarction Onset of illness Orthopedics Outcome Study Oxidative Stress Pathologic Pathology Patients Perfusion Phenotype Physiological Physiology Prevalence Proteins Proteoglycan Reactive Oxygen Species Recovery Research Risk Role Serum Stress Stroke System Testing Tissue Engineering United States Valine Vasodilation Western Blotting Woman amino acid metabolism atherogenesis atherosclerosis risk cardiovascular risk factor cell growth chondroitin sulfate glycosaminoglycan cytokine disability disease phenotype endothelial dysfunction fabrication human tissue in vitro Model interest low and middle-income countries member metabolomics monocyte mouse model negative affect neonatal human novel novel therapeutics oxidized low density lipoprotein polysulfated glycosaminoglycan prevent therapeutic target vascular inflammation

项目摘要

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.

支链氨基酸 (BCAA) 亮氨酸、异亮氨酸和缬氨酸水平升高高度相关随着CVD和不良心血管事件（例如心脏病发作）的发展。一些机制 BCAA、代谢疾病和心力衰竭之间的联系已有记录。然而，目前尚不清楚是否 BCAA 直接与血管细胞相互作用，促进 CVD 的发生和进展。鼠标型号有用于研究 CVD 和 BCAA 的人类生理学应用均存在局限性。目前的动脉粥样硬化体外模型仅限于动脉粥样硬化形成的早期阶段，并且具有挑战性概括更高级的动脉粥样硬化变化。为了解决这个问题，我们将开发一个模型使用人体组织工程血管系统 (TEBV) 模型进行中间动脉粥样硬化病变疾病病理学富含糖胺聚糖硫酸软骨素 (CS) 的胶原蛋白细胞外基质，用修饰的低密度脂蛋白（LDL）治疗。我们将使用经过修饰的 LDL 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BCAA机制的平台。 TEBV模型由胶原蛋白或CS-胶原蛋白血管与封装的人新生儿真皮制成成纤维细胞作为血管壁的内侧细胞和内皮细胞的内皮化内腔集落形成细胞（ECFC）。以 2 mL/分钟的速度灌注培养基，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，并证明血管活性功能障碍增加与胶原 TEBV 相比，内皮-白细胞相互作用。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，用升高的支链氨基酸和氧化的低- 密度脂蛋白导致线粒体氧化应激增加，以及 LC3B 表达减少，一种自噬体蛋白。这诱发了 TEBV 系统的早期动脉粥样硬化事件：受损内皮控制的血管舒张和白细胞对内皮的粘附。自噬通量将进一步通过Western Blot测定LC3-I至LC3-II的转化。血管细胞，尤其是内皮细胞，可能对升高的 BCAA 的影响敏感，因为它们对 BCAA 代谢没有显着贡献，导致细胞内代谢物的积累和线粒体应激。这将通过代谢组学进行测试分析血管细胞中的支链氨基酸及其下游代谢物。使用 TEBV，我们将确定 BCAA 在早期和中期动脉粥样硬化中的作用。在研究结束时，我们将得到一个动脉粥样硬化的中间病变模型，支链氨基酸与心血管事件的联系机制变得更加清晰，并且将确定动脉粥样硬化的潜在治疗靶点。