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

• 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
• 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

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与 随着心血管疾病和不良心血管事件的发展，如心脏病发作。一些机制 与支链氨基酸、代谢性疾病和心力衰竭有关。然而，目前尚不清楚， 支链氨基酸直接与血管细胞相互作用，使CVD发病和进展。小鼠模型， 被开发用于研究CVD和BCAA两者在其对人类生理学的适用性方面都具有局限性。 目前动脉粥样硬化的体外模型限于动脉粥样硬化形成的早期阶段，并且具有挑战性， 概括了更严重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， 使用人组织工程血管系统（TEBV）模型， 富含糖胺聚糖硫酸软骨素（CS）的疾病病理学胶原细胞外基质， 用修饰的低密度脂蛋白（LDL）治疗。我们将使用经修饰的LDL处理的胶原TEBV作为 早期动脉粥样硬化模型。这些平台将被用作研究BCAA在CVD中的机制的平台。 TEBV模型由胶原或CS-胶原血管与包被的人新生儿真皮制成 成纤维细胞充当血管壁中的中膜细胞和内皮化的内皮细胞内腔。 集落形成细胞（ECFC）。以2 mL/min的速率灌注培养基，可培养至 6周我们已经证明，CS-富集血管具有增强的敏感性， 修饰的LDL的炎症作用，并证明增加的血管血管活性功能障碍， 与胶原TEBV相比，内皮-白细胞相互作用。我们还证明， 生理环境中的BCAA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BCAA和氧化的低- 密度脂蛋白引起线粒体氧化应激增加，以及LC 3B表达减少， 自噬体蛋白。这在TEBV系统中诱导了早期动脉粥样硬化事件： 内皮控制的血管舒张和白细胞粘附于内皮。自噬通量将进一步 通过蛋白质印迹分析LC 3-I至LC 3-II的转化。血管细胞，特别是内皮细胞，可能是 对BCAA升高的影响敏感，因为它们对BCAA代谢没有显著贡献， 导致代谢物和线粒体应激的细胞内积累。这将通过代谢组学进行检测 血管细胞中支链氨基酸及其下游代谢产物的分析。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BCAA与心血管事件联系起来的机制将 将变得更加清晰，并将确定动脉粥样硬化的潜在治疗靶点。