Regulation of Lymphangiogenesis by Thrombospondin 1

血小板反应蛋白 1 对淋巴管生成的调节

• 批准号: 10516406
• 负责人: Bhupesh Singla
• 金额: $ 24.52万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10516406
• 关键词: Regulation; Lymphangiogenesis; Thrombospondin

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.

在美国，动脉粥样硬化占死亡的大多数，其特征是动脉壁中脂质过度积聚。动脉脂质蓄积是由于细胞脂蛋白摄取、胆固醇清除和细胞脂质重新合成失衡所致。淋巴网络是从动脉粥样硬化病变中清除胆固醇和炎性细胞/细胞因子的不可或缺的参与者。淋巴引流的手术和遗传破坏导致动脉粥样硬化病变形成加重。然而，动脉粥样硬化血管中调节淋巴管生成的确切内源性因素和机制仍不清楚。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疾病联系起来，然而TSP 1促进动脉粥样硬化病变形成的机制及其对动脉淋巴管生成的影响仍然未知。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）在病理生理相关浓度的TSP 1抑制体外淋巴管生成;和iii）高脂血症小鼠中TSP 1和CD 47的整体缺失减轻动脉粥样硬化。基于这些结果，我推测TSP 1通过CD 47依赖性机制损害淋巴管生成，通过LEC CD 47缺失诱导淋巴管生成增加胆固醇逆向转运，减少动脉粥样硬化的发展。该提案将在体外检查LEC中TSP 1介导的CD 47活化是否通过增加ROS产生、降低NO生物利用度和减弱PI 3 K-Akt和ERK 1/2活化来抑制淋巴管生成（目的1）。该提案将研究是否在LEC中特异性抑制CD 47信号传导增加淋巴管生成，改善胆固醇逆向转运（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敲除小鼠来验证该假设。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。这笔赠款将是至关重要的博士. Singla实现以下短期和长期目标：1）获得额外的科学培训方法和概念; 2）合并尚未独特的基质蛋白生物学和生物化学领域的目标，朝着开辟新的发现途径; 3）建立博士. Singla的独立研究计划; 4）发表高影响力的通讯作者文章，并开发具有高度竞争力的R 01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。