Regulation of Lymphangiogenesis by Thrombospondin 1

项目摘要 在美国，动脉粥样硬化占死亡人数的大多数，其特征是脂质过多 在动脉壁中积聚。动脉脂质蓄积是细胞脂蛋白失衡的结果 摄取、胆固醇去除和细胞脂质从头合成。淋巴网络是一个不可或缺的 参与从动脉粥样硬化病变中清除胆固醇和炎性细胞/细胞因子。手术和 淋巴引流的遗传破坏导致动脉粥样硬化损伤形成加重。但 动脉粥样硬化血管中调节淋巴管生成的精确内源性因子和机制仍然存在 身份不明先前的研究已经将基质蛋白血小板反应蛋白-1（TSP-1）水平升高与人类 动脉粥样硬化性疾病，然而，TSP 1促进动脉粥样硬化病变形成的机制 并且其对动脉淋巴管生成的作用仍然未知。我们的初步研究表明，i） 淋巴管内皮细胞（LEC）表达高水平的TSP 1受体，CD 47; ii）TSP 1在病理生理 相关浓度在体外抑制淋巴管生成;和iii）TSP 1和CD 47的整体缺失， 高脂血症小鼠减弱动脉粥样硬化。基于这些结果，我假设TSP 1损害了 通过CD 47依赖性机制的淋巴管生成和通过LEC CD 47诱导淋巴管生成 缺失增加胆固醇逆向转运并减少动脉粥样硬化的发展。该提案将 体外检测LEC中TSP 1介导的CD 47活化是否通过增加ROS抑制淋巴管生成 产生，降低NO生物利用度和减弱PI 3 K-Akt和ERK 1/2激活（目的1）。的 该提案将研究特异性抑制LEC中的CD 47信号传导是否会增加淋巴管生成， 改善胆固醇逆向转运（目的2）和抑制体内动脉粥样硬化的发展（目的3）。 各种分子生物学和成像技术，ROS/NO检测方法，淋巴管生成测定， PCSK 9-AAV 8诱导的动脉粥样硬化模型，体内胆固醇逆向转运，淋巴功能分析 和LEC特异性CD 47敲除小鼠将被用于测试该假设。成功完成 拟议的研究将有助于更好地了解淋巴管生成和淋巴管生成的调节。 动脉粥样硬化中的血管功能。这笔赠款将是至关重要的博士。Singla实现以下短期和长期- 学期目标：1）在方法和概念上获得额外的科学培训; 2）合并 然而，不同的基质蛋白生物学和生物学领域的目标是开辟新的途径， 发现; 3）建立Singla博士的独立研究计划; 4）发表高影响力的相应研究成果。 撰写文章并开发极具竞争力的R 01资助申请。辛格拉博士已经组织了一个 多学科团队，包括他的导师，顾问和顾问，以指导他的职业生涯， 独立性，并协助完成拟议的研究。

Project Summary Atherosclerosis accounts for the majority of deaths in the United States and is characterized by excessive lipid accumulation in the arterial wall. Arterial lipid accumulation results from the imbalance of cellular lipoprotein uptake, removal of cholesterol and de novo cellular lipid synthesis. The lymphatic network is an indispensable player in the removal of cholesterol and inflammatory cells/cytokines from atherosclerotic lesions. Surgical and genetic disruption of lymphatic drainage leads to aggravated atherosclerotic lesion formation. However, the precise endogenous factors and mechanisms regulating lymphangiogenesis in atherosclerotic vessels remain unidentified. Previous studies have linked elevated matrix protein thrombospondin-1 (TSP1) levels with human atherosclerotic disease, however the mechanisms by which TSP1 contributes to atherosclerotic lesion formation and its effect on arterial lymphangiogenesis remain unknown. Our preliminary studies demonstrate that i) lymphatic endothelial cells (LEC) express high levels of TSP1 receptor, CD47; ii) TSP1 at pathophysiologically relevant concentrations inhibits lymphangiogenesis in vitro; and iii) global deletion of TSP1 and CD47 in hypercholesteremic mice attenuates atherosclerosis. Based on these results, I hypothesize that TSP1 impairs lymphangiogenesis via CD47-dependent mechanisms and induction of lymphangiogenesis via LEC CD47 deletion increases reverse cholesterol transport and decreases atherosclerosis development. The proposal will examine in vitro whether TSP1-mediated CD47 activation in LEC inhibits lymphangiogenesis via increased ROS generation, decreased NO bioavailability and attenuation of PI3K-Akt and ERK1/2 activation (Aim 1). The proposal will investigate whether inhibiting CD47 signaling specifically in LEC increases lymphangiogenesis, improves reverse cholesterol transport (Aim 2) and suppresses atherosclerosis development in vivo (Aim 3). Various molecular biology and imaging techniques, ROS/NO detection methods, lymphangiogenesis assays, PCSK9-AAV8-induced atherosclerosis model, in vivo reverse cholesterol transport, lymphatic function analysis and LEC-specific CD47 knockout mice will be employed to test the hypothesis. Successful completion of the proposed studies will contribute to a better understanding of the regulation of lymphangiogenesis and lymphatic vessel function in atherosclerosis. This grant will be critical for Dr. Singla to achieve the following short- and long- term objectives: 1) to acquire additional scientific training both methodologically and conceptually; 2) to merge the yet distinct matrix protein biology and lymphatics fields with the goals toward opening up new avenues of discovery; 3) to establish Dr. Singla’s independent research program; 4) publish high-impact corresponding author articles and develop a highly competitive R01 grant application. Dr. Singla has assembled a multidisciplinary team, including his mentors, consultants, and advisors to guide his career towards independence and assist with the completion of the proposed research study.