Regulation of Lymphangiogenesis by Thrombospondin 1

在美国，动脉粥样硬化是导致死亡的主要原因，其特征是动脉壁的脂质过度积聚。动脉脂质积累是由于细胞脂蛋白摄取、胆固醇去除和新生细胞脂质合成的不平衡造成的。淋巴网络在清除动脉粥样硬化病变中的胆固醇和炎症细胞/细胞因子中起着不可或缺的作用。手术和遗传破坏淋巴引流导致加重动脉粥样硬化病变的形成。然而，调节动脉粥样硬化血管中淋巴管生成的确切内源性因素和机制尚不清楚。先前的研究已将基质蛋白血栓反应蛋白-1 （TSP1）水平升高与人类动脉粥样硬化疾病联系起来，然而TSP1促进动脉粥样硬化病变形成及其对动脉淋巴管生成的影响的机制尚不清楚。我们的初步研究表明，i)淋巴内皮细胞（LEC）表达高水平的TSP1受体CD47；ii)病理生理相关浓度的TSP1在体外抑制淋巴管生成；iii) TSP1和CD47在高胆固醇小鼠中的整体缺失可减轻动脉粥样硬化。基于这些结果，我假设TSP1通过CD47依赖机制损害淋巴管生成，通过LEC CD47缺失诱导淋巴管生成增加逆向胆固醇运输并减少动脉粥样硬化的发展。该提案将在体外研究tsp1介导的LEC中CD47激活是否通过增加ROS生成、降低NO生物利用度和抑制PI3K-Akt和ERK1/2激活来抑制淋巴管生成（Aim 1）。该提案将研究在LEC中特异性抑制CD47信号是否会增加淋巴管生成，改善胆固醇逆向运输（Aim 2）并抑制体内动脉粥样硬化的发展（Aim 3）。将采用各种分子生物学和影像学技术、ROS/NO检测方法、淋巴管生成实验、pcsk9 - aav8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CD47敲除小鼠来验证这一假设。这些研究的成功完成将有助于更好地理解动脉粥样硬化中淋巴管生成和淋巴管功能的调节。这笔拨款将对辛格拉博士实现以下短期和长期目标至关重要：1)在方法和概念上获得额外的科学培训；2)将基质蛋白生物学和淋巴学领域结合起来，以开辟新的发现途径；3)建立辛格拉博士的独立研究项目；4)发表具有高影响力的通讯作者文章，并提出竞争激烈的R01资助申请。Singla博士组建了一个多学科团队，包括他的导师、顾问和顾问，以指导他的职业生涯走向独立，并协助完成拟议的研究。

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.