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，其特征是动脉壁内脂质过度堆积。动脉脂肪积聚是细胞内脂蛋白摄取、胆固醇排出和细胞内脂肪合成失衡的结果。淋巴管网络在清除动脉粥样硬化病变中的胆固醇和炎性细胞/细胞因子方面起着不可或缺的作用。淋巴管引流的外科和遗传性中断会导致动脉粥样硬化病变的加重。然而，调控动脉粥样硬化血管淋巴管生成的确切内源性因素和机制仍不清楚。以往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性疾病联系起来，但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：1)淋巴管内皮细胞(LEC)表达高水平的TSP1受体CD47；2)病理生理相关浓度的TSP1可抑制体外淋巴管的生成；3)高胆固醇血症小鼠体内TSP1和CD47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依赖的机制损害淋巴管生成，并通过LEC CD47缺失诱导淋巴管生成增加胆固醇的反向运输，减少动脉粥样硬化的发展。该提案将在体外检测TSP1介导的CD47在LEC中的激活是否通过增加ROS生成、降低NO的生物利用度以及减弱PI3K-Akt和ERK1/2的激活来抑制淋巴管生成(目标1)。该提案将调查在LEC中抑制CD47信号是否能增加淋巴管的生成，改善胆固醇的反向运输(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、淋巴功能分析和LEC特异性CD47基因敲除小鼠将被用于验证这一假说。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。这笔赠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更多的科学培训；2)将独特的基质蛋白质生物学和淋巴学领域与开辟新的发现途径的目标结合起来；3)建立辛格拉博士的独立研究计划；4)发表高影响力的通信作者文章，并开发具有极强竞争力的R01拨款申请。辛格拉博士已经组建了一个多学科团队，其中包括他的导师、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.