Regulation of vascular smooth muscle cell function in atherosclerosis

血管平滑肌细胞（VSMC）的表型和功能调控在血管平滑肌细胞的形成和发展中起着重要的作用 在动脉粥样硬化发病机制中的作用。血管钙化是动脉粥样硬化的特征性表现 预测动脉粥样硬化患者的不良心血管结局。在过去的二十年里， 研究表明，血管钙化是一个受调节的过程;成骨分化和 VSMC的钙化对血管钙化的发展有重要作用。钙信号是 对调节VSMC功能至关重要。然而，关键的细胞内钙信号传导介质的作用， 磷脂酶Cγ（PLCγ）对VSMC钙化的调节作用尚不清楚。我们的初步研究 表明PLCγ2的缺失显著增加了VSMC和动脉粥样硬化病变的钙化 ApoE-/-小鼠利用PLCγ2缺失小鼠（PLCγ2-/-）的VSMC，我们测定了PLCγ2对VSMC增殖的直接影响， 缺乏促进VSMC钙化，这是独立的PLCγ1亚型或已知的PLCγ- 介导的信号通路，支持PLCγ2在调节VSMC中的独特和新颖的功能 钙化PLCγ2基因缺失改变了VSMC的细胞骨架结构，增加了基质囊泡的分泌 (MVs)含有钙和基质蛋白的膜结合纳米颗粒。MV分泌是一个关键的细胞 在骨形成过程中引发细胞外基质钙化的骨生成事件;最近 在血管钙化中起重要作用。然而，MV的分子调控 对VSMC的分泌知之甚少。我们发现，恢复PLCγ2使MV分泌正常化， 抑制PLCγ2-/- VSMC钙化。此外，PLCγ2与膜相关的 丝状蛋白，septin 4/5，已显示与膜磷脂动态相互作用 以及调节细胞骨架排列和突触小泡分泌的胞吐机制蛋白质。的 septins在VSMC MV分泌中的作用尚不清楚，我们发现PLCγ2/septin 4/5相互作用， Septin 4/5在PLCγ2-/- VSMC MV中的表达增加支持Septin在VSMC MV调节中的新作用 分泌物和钙化。因此，我们推测PLCγ2缺陷可诱导VSMC钙化， 通过改变的隔蛋白/肌动蛋白-细胞骨架结构，导致MV分泌增加。选用我们的 目的1在小鼠体内研究SMC特异性PLCγ2在调节血管功能中的作用; 目的2阐明PLCγ2调控VSMC钙化的分子机制。这些 研究将阐明SMC衍生的PLCγ2在调节细胞骨架结构和MV中的整合作用。 分泌导致VSMC钙化。人类PLCγ2突变最近被确定为导致 免疫性疾病，但其潜在机制尚未完全了解。因此， PLCγ2调节血管功能的新功能和机制应提供新的见解， PLCγ2在血管系统以外的健康和疾病中的综合作用。

ABSTRACT Regulation of vascular smooth muscle cells (VSMC) phenotype and function plays important roles in the pathogenesis of atherosclerosis. Vascular calcification is a characteristic feature of atherosclerosis that predicts adverse cardiovascular outcome of atherosclerotic patients. Over the last two decades, increasing studies have demonstrated that vascular calcification is a regulated process; and osteogenic differentiation and calcification of VSMC contributes significantly to the development of vascular calcification. Calcium signaling is critical in regulating VSMC function. However, the role of the key intracellular calcium signaling mediator, phospholipase Cγ (PLCγ), in regulating VSMC calcification is entirely unknown. Our preliminary studies demonstrated that deletion of PLCγ2 markedly increased calcification in VSMC and in atherosclerotic lesions of ApoE-/- mice. Using VSMC from PLCγ2 deletion mice (PLCγ2-/-), we determined a direct effect of PLCγ2 deficiency on promoting VSMC calcification, which was independent of the PLCγ1 isoform or the known PLCγ- mediated signaling pathways, supporting a unique and novel function of PLCγ2 in regulating VSMC calcification. PLCγ2 deletion in VSMC altered cytoskeleton structure and increased secretion of matrix vesicles (MVs), membrane-bound nanoparticles that harbor calcium and matrix proteins. MV secretion is a key cellular event in osteogenesis that initiates extracellular matrix calcification during bone formation; and has recently been shown to play an important role in vascular calcification. However, the molecular regulations of MV secretion in VSMC are poorly understood. We found that restoring PLCγ2 normalized MV secretion and inhibited calcification of the PLCγ2-/- VSMC. Furthermore, PLCγ2 interacted with membrane-associated filamentous proteins, septin 4/5, which have been shown to dynamically interact with membrane phospholipids and exocytosis machinery proteins that regulate cytoskeleton arrangement and synaptic vesicle secretion. The roles of septins in VSMC MV secretion are unknown, our findings of PLCγ2/septin4/5 interactions and increased septin 4/5 in MVs from PLCγ2-/- VSMC support a new role of septins in regulating VSMC MV secretion and calcification. Therefore, we hypothesize that PLCγ2 deficiency induces VSMC calcification via altered septin/actin-cytoskeleton structure that leads to increased MV secretion. With our new mouse models, Aim 1 will determine the function of SMC-specific PLCγ2 in regulating vascular function in vivo; and Aim 2 will elucidate the molecular mechanisms underlying PLCγ2-regulated VSMC calcification. These studies will elucidate an integrative role of SMC-derived PLCγ2 in regulating cytoskeleton structure and MV secretion that lead to VSMC calcification. PLCγ2 mutations in humans have recently been identified to cause immunological diseases but the underlying mechanisms are not fully understood. Therefore, delineating the novel function and mechanisms whereby PLCγ2-regulated vascular function should provide novel insights into the comprehensive function of PLCγ2 in health and disease beyond the vascular system.