Regulation of Lymphangiogenesis by Thrombospondin 1

项目摘要 动脉粥样硬化是美国大多数人死亡的原因，其特点是血脂过高 在动脉壁内堆积。细胞脂蛋白失衡导致动脉脂肪堆积 摄取、清除胆固醇和从头开始细胞脂肪合成。淋巴网络是不可或缺的 在清除动脉粥样硬化病变中的胆固醇和炎性细胞/细胞因子方面发挥了重要作用。外科手术和 淋巴引流的遗传障碍会导致动脉粥样硬化病变的加重。然而， 动脉粥样硬化血管中调控淋巴管生成的精确内源性因素和机制仍然存在 身份不明。以前的研究已经将基质蛋白凝血酶反应蛋白-1(TSP1)水平升高与人类 动脉粥样硬化性疾病，然而，TSP1促进动脉粥样硬化病变形成的机制 其对动脉淋巴管生成的影响尚不清楚。我们的初步研究表明，i) 淋巴管内皮细胞(LEC)高水平表达TSP1受体CD47；II)TSP1在病理生理上的表达 相关浓度在体外抑制淋巴管生成；以及iii)TSP1和CD47的整体缺失 高胆固醇血症小鼠可减轻动脉粥样硬化。基于这些结果，我假设TSP1会损害 CD47依赖的淋巴管生成机制和LEC CD47诱导的淋巴管生成 缺失增加了胆固醇的反向运输，减少了动脉粥样硬化的发展。这项提议将 体外检测TSP1介导的CD47激活LEC是否通过增加ROS抑制淋巴管生成 产生，降低NO的生物利用度和抑制PI3K-Akt和ERK1/2的激活(目标1)。这个 该提案将调查在LEC中特异性抑制CD47信号是否会增加淋巴管生成， 改善体内胆固醇的反向转运(目标2)和抑制体内动脉粥样硬化的发展(目标3)。 各种分子生物学和成像技术，ROS/NO检测方法，淋巴管生成分析， PCSK9-AAV8诱导的动脉粥样硬化模型、体内胆固醇反向转运、淋巴功能分析 而LEC特异性CD47基因敲除小鼠将被用来检验这一假设。圆满完成 拟议的研究将有助于更好地理解淋巴管生成和淋巴管的调节。 动脉粥样硬化中的血管功能。这笔赠款对辛格拉博士实现以下短期和长期目标至关重要-- 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，独特的基质蛋白生物学和淋巴学领域的目标是开辟新的途径 发现；3)建立辛格拉博士的独立研究计划；4)发表高影响力的相应 撰写文章并开发一个极具竞争力的R01拨款申请。辛格拉博士已经组装了一个 多学科团队，包括他的导师、顾问和顾问，指导他的职业生涯 独立并协助完成拟议的研究研究。

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.