Molecular Mechanisms Governing Vascular Cell Function and Phenotype in Health and Disease

项目摘要/摘要 慢性炎症和循环胆固醇升高导致的内皮功能障碍促进 导致冠心病的主要动脉内皮下斑块的形成 世界范围内致病和死亡的原因。损伤内皮细胞的修复有望治疗 然而，内源性内皮细胞(EC)再生是一个低效的过程。有能力 恢复动脉内皮的通畅将提供一个显著的治疗进步。因为 血管平滑肌细胞(VSMCs)构成了动脉壁中的大多数细胞，并能够 表型可塑性对病理生理刺激的反应，这些细胞代表了一个吸引人的来源 有功能的内皮细胞。解开控制反式转运蛋白的分子机制和信号通路 将血管平滑肌细胞分化为内皮细胞修复损伤的内皮细胞将建立一种新的治疗模式 治疗冠心病。我们的长期目标是发现新的分子和信号通路， 促进VSMC向内皮细胞的转化(MEndoT)。我们的实验室已经鉴定并鉴定了一个家族 进化上保守的内吞适配器蛋白称为epsins，它在协调中起着至关重要的作用 内吞作用和信号转导。我们的研究表明，内皮细胞和髓样细胞中epsins 1和2的丢失 减少血管炎症，防止斑块的形成和发展。为了进一步评估 靶向促病变进展的细胞内的epsins的治疗作用以及斑块组成和 稳定性，我们将使用最近创建的具有VSMC特异性缺陷的疾病特异性小鼠 伊普斯。我们建议询问VSMC epsin蛋白在这些过程中的功能，并建立 这些蛋白的治疗靶向将促进有益的VSMC表型转换。到目前为止，我们 初步研究表明，VSMC epins缺陷的ApoE-/-小鼠的 斑块大小、增强的斑块稳定性(包括增加纤维帽面积和ACTA2细胞 CaP)，减少浸润性细胞(CD45免疫和炎性细胞以及CD68泡沫 细胞)，血管僵硬和钙化显著减少。此外，rna-seq分析显示 KLF4，控制VSMC表型转换的多潜能转录因子，被下调 内皮蛋白丢失，以及oxLDL触发的Runx2泛素化和降解。根据这些调查结果，我们将 使用独特的突变小鼠、体外模型和新试剂研究以下特定目标：1) 确定内皮肽调节表型转换和间充质转化的分子机制 血管内皮细胞分化，2)确定内皮肽调节VSMC的分子机制 成骨和促进动脉僵硬，以及3)确定靶向内啡肽的治疗潜力 用于动脉粥样硬化的形成和消退。如果成果丰硕，拟议的研究将补充我们先前的工作，并 强化epsin蛋白可作为治疗冠心病的有效靶点的概念。

PROJECT SUMMARY/ABSTRACT Endothelial dysfunction resulting from chronic inflammation and elevated circulating cholesterol promotes the formation of plaques in the sub-endothelium of major arteries causing coronary heart disease—a leading cause of morbidity and mortality worldwide. Repair of the injured endothelium holds great promise to treat heart disease; however, endogenous endothelial cell (EC) regeneration is an inefficient process. The ability to restore patency of the arterial endothelium would provide a significant therapeutic advancement. Because vascular smooth muscle cells (VSMCs) constitute the majority of cells in the arterial wall and are capable of phenotypic plasticity in response to pathophysiological stimuli, these cells represent an appealing source of functional endothelial cells. Unraveling the molecular mechanisms and signaling pathways that govern trans- differentiation of VSMCs into ECs to mend the injured endothelium would establish a novel treatment paradigm for coronary heart disease. Our long-term goal is to discover new molecules and signaling pathways that facilitate VSMC-to-endothelial transition (MEndoT). Our laboratory has identified and characterized a family of evolutionarily-conserved endocytic adaptor proteins called epsins, which have crucial roles in coordinating endocytosis and signal transduction. Our studies show that loss of epsins 1 and 2 in ECs and myeloid cells reduces vascular inflammation and prevents plaque initiation and progression. To further assess the therapeutic effects of targeting epsins in cells that drive lesion progression as well as plaque composition and stability, we will use recently created disease-specific mice harboring VSMC-specific deficiency of these epsins. We propose to interrogate the function of VSMC epsin proteins in these processes and establish that therapeutic targeting of these proteins will promote beneficial VSMC phenotype switching. So far, our preliminary studies indicate that ApoE-/- mice with a deficiency in VSMC epsins have a significant reduction in plaque size, enhanced plaque stability (including an increase in fibrous cap area and ACTA2+ cells within the cap), a reduction in the number of infiltrating cells (CD45+ immune and inflammatory cells and CD68+ foam cells), and a prominent decrease in vascular stiffness and calcification. In addition, RNA-seq analyses show that Klf4, the pluripotent transcriptional factor controlling phenotypic switching of VSMCs, is downregulated by epsin loss, as is oxLDL-triggered Runx2 ubiquitination and degradation. In light of these findings, we will investigate the following Specific Aims using unique mutant mice, in vitro models, and novel reagents: 1) To determine the molecular mechanisms by which epsins regulate phenotype switching and mesenchymal-to- endothelial differentiation, 2) To determine the molecular mechanisms by which epsins regulate VSMC osteogenesis and promote arterial stiffness, and 3) To determine the therapeutic potential of targeting epsins for atheroma formation and resolution. If fruitful, the proposed study will complement our prior work and strengthen the concept that epsin proteins may serve as a potent therapeutic target for coronary heart disease.