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

项目总结/摘要 由慢性炎症和循环胆固醇升高引起的内皮功能障碍促进了内皮细胞的增殖。 在主要动脉内皮下形成斑块导致冠心病-一个主要的 全世界发病率和死亡率原因。修复受损的内皮细胞有望治疗 然而，内源性内皮细胞（EC）再生是一个低效的过程。的能力 恢复动脉内皮的开放性将提供显著的治疗进步。因为 血管平滑肌细胞（VSMC）构成动脉壁中的大多数细胞， 表型可塑性响应病理生理刺激，这些细胞代表了一个有吸引力的来源， 功能性内皮细胞揭示了控制跨- 将VSMCs分化为EC以修复受损的内皮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， 促进血管平滑肌细胞向内皮细胞转化（MEEndoT）。我们的实验室已经鉴定并鉴定了一个家族， 进化上保守的内吞衔接蛋白称为epsins，在协调 内吞作用和信号转导。我们的研究表明，内皮细胞和髓系细胞中epsin 1和2的缺失 减少血管炎症并防止斑块形成和发展。以进一步评估 靶向驱动病变进展的细胞中的epsin以及斑块组成的治疗效果， 稳定性，我们将使用最近创建的疾病特异性小鼠，这些小鼠具有VSMC特异性缺陷， 胰蛋白酶我们建议询问VSMC epsin蛋白在这些过程中的功能，并建立 这些蛋白质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 epsins缺乏的ApoE-/-小鼠， 斑块大小、增强的斑块稳定性（包括纤维帽面积和ACTA 2+细胞的增加）， cap）、浸润细胞（CD 45+免疫和炎性细胞以及CD 68+泡沫细胞）数量的减少 细胞），以及血管硬度和钙化的显著降低。此外，RNA-seq分析显示， Klf 4是控制VSMC表型转换的多能转录因子， 胰蛋白酶损失，如oxLDL触发的Runx 2泛素化和降解。根据这些发现，我们将 使用独特的突变小鼠、体外模型和新型试剂研究以下特定目的：1） 确定epsins调节表型转换和间充质-间充质转化的分子机制。 2）确定epsins调节VSMC的分子机制 骨生成和促进动脉硬化，以及3）确定靶向epsin的治疗潜力 用于动脉粥样硬化形成和消退。如果富有成果，拟议的研究将补充我们之前的工作， 加强epsin蛋白可能作为冠心病有效治疗靶点的概念。

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.