Novel genetic Insight into the molecular pathogenesis of atherosclerosis

动脉粥样硬化是一种慢性炎症性疾病，其特征是由胆固醇组成的动脉斑块， 填充的巨噬细胞、坏死的脂质核心和含有血管平滑肌细胞（SMC）的纤维帽。 广泛的工作已经确定了遗传变异如何改变脂质水平和炎症， 动脉粥样硬化，但较少知道如何影响SMC的遗传改变易患动脉粥样硬化。 动脉粥样硬化斑块中平滑肌细胞丢失，这一发现使平滑肌细胞在动脉粥样硬化病变中的作用扩大 分化标志物并启动巨噬细胞标志物的表达，从而成为巨噬细胞样细胞。 类似地，SMC在体外下调SMC标志物，但在暴露于 游离胆固醇我们确定了ACTA 2中的一些杂合错义突变，ACTA 2编码 SMC特异性α-肌动蛋白亚型，使个体易患胸主动脉疾病和早发性 冠状动脉疾病（CAD）。我们已经设计了一个具有这样一个突变和SMC的小鼠模型， 从Acta 2 R149 C/+中分离出的巨噬细胞去分化并在很大程度上增加巨噬细胞标志物的表达 游离胆固醇浓度低于野生型SMC。此外，当Acta 2 R149 C/+小鼠被 与Apoe-/-小鼠杂交并喂食高脂肪饮食，双突变小鼠具有显著增加的负担， 动脉粥样硬化斑块的发生率。我们假设早发性 与SM α-肌动蛋白R149 C突变相关的CAD是由于突变肌动蛋白折叠破坏，导致 增加Klf 4活化并增强SMC向巨噬细胞样细胞的表型转换。我们进一步 据推测，尽管许多ACTA 2突变可能增加SMC增殖和迁移，但只有ACTA 2的变异 增加SMC转换为巨噬细胞样细胞将易患早发性CAD。目标是解决 这些假设如下：（1）表征Acta 2 R149 C/+小鼠中的动脉粥样硬化病变， 包括确定病变中SMC和巨噬细胞的来源;（2）确定细胞通路 负责增强的Acta 2 R149 C/+ SMC转换为巨噬细胞样细胞与胆固醇接触， 并通过遗传学方法评估这些途径在Acta 2 R149 C/+小鼠中增加斑块负荷中的作用。 操纵(3)确定CAD相关ACTA 2突变如何破坏伴侣蛋白CCT的折叠 复杂，并显示SM α-肌动蛋白折叠缺陷和CAD易感ACTA 2错义之间的因果关系 突变因此，拟议的研究将提供有价值的新见解SMC表型的作用， 转换为动脉粥样硬化的危险因素，并且还可以确定新的治疗靶点， 甚至阻止疾病发展。

Atherosclerosis is a chronic inflammatory condition characterized by arterial plaques composed of cholesterol- filled macrophages, a necrotic lipid core, and a fibrous cap containing vascular smooth muscle cells (SMCs). Extensive work has defined how genetic variants altering lipid levels and inflammation contribute to atherosclerosis, but less is known as to how genetic alterations affecting SMCs predispose to atherosclerosis. An expanded role for SMCs in atherosclerotic lesions is evoked by the finding that SMCs in plaques lose SMC differentiation markers and initiate expression of macrophage markers, thus becoming a macrophage-like cell. Similarly, SMCs downregulate SMC markers in vitro, but upregulate macrophage markers with exposure to free cholesterol. We determined that some heterozygous missense mutations in ACTA2, which encodes the SMC-specific isoform of α-actin, predispose individuals to both thoracic aortic disease and early onset coronary artery disease (CAD). We have engineered a mouse model with one such mutation and SMCs explanted from Acta2R149C/+ aortas de-differentiate and increase expression of macrophage markers at much lower concentrations of free cholesterol than wildtype SMCs. Furthermore, when the Acta2R149C/+ mice are crossed into Apoe-/- mice and fed a high fat diet, the double mutant mice have a significantly increased burden of atherosclerotic plaques when compared to similarly treated Apoe-/- mice. We hypothesize that early onset CAD associated with the SM α-actin R149C mutation is due to disrupted folding of the mutant actin, leading to increased Klf4 activation and augmented SMC phenotypic switching to macrophage-like cells. We further speculate that, although many ACTA2 mutations may increase SMC proliferation and migration, only variants that increase SMC switching to macrophage-like cells will predispose to early onset CAD. The aims to address these hypotheses are the following: (1) Characterize the atherosclerotic lesions in the Acta2R149C/+ mice, including identifying the origin of the SMCs and macrophages in the lesions; (2) Identify cellular pathways responsible for enhanced Acta2R149C/+ SMC switching to a macrophage-like cell with exposure to cholesterol, and assess the role of these pathways in the increased plaque burden in Acta2R149C/+ mice through genetic manipulation. (3) Determine how CAD-associated ACTA2 mutations disrupt folding by the chaperonin CCT complex, and show causality between SM α-actin folding defects and CAD-predisposing ACTA2 missense mutations. Thus, the proposed research will provide valuable new insights into the role of SMC phenotypic switching as a risk factor for atherosclerosis, and may also identify novel therapeutic targets that can delay or even prevent disease progression.