Regulation of vascular smooth muscle cell function in atherosclerosis

摘要血管平滑肌细胞(VSMC)的表型和功能调节非常重要。 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 这预示着动脉粥样硬化患者的不良心血管后果。在过去的二十年里，越来越多的人 研究表明，血管钙化是一个受调节的过程；成骨分化和 VSMC的钙化在血管钙化的发展中起着重要作用。钙信号是 在调节VSMC功能中起关键作用。然而，关键的细胞内钙信号媒介的作用， 磷脂酶Cγ(PLCγ)在调节血管平滑肌细胞钙化中的作用尚不清楚。我们的初步研究 研究表明，PLCγ2的缺失显著增加了血管平滑肌细胞和动脉粥样硬化病变的钙化 载脂蛋白E-/-小鼠。利用PLCγ2缺失小鼠的血管平滑肌细胞(PLCγ2-/-)，我们确定了PLCγ2的直接作用 在促进血管平滑肌细胞钙化方面的不足，这不依赖于PLCγ1亚型或已知的PLCγ- 介导的信号通路支持PLCγ2独特而新颖的调节血管平滑肌细胞的功能 钙化。血管平滑肌细胞中PLCγ2缺失改变细胞骨架结构并增加基质囊泡的分泌 (MVS)是一种膜结合纳米颗粒，含有钙和基质蛋白。MV的分泌是一个关键的细胞 在骨形成过程中启动细胞外基质钙化的成骨事件；最近 已被证明在血管钙化中起着重要作用。然而，MV的分子调控 人们对VSMC的分泌知之甚少。我们发现，恢复PLCγ2使MV分泌正常化，并 抑制γ2-/-VSMC的钙化。此外，PLCγ2与膜相关蛋白相互作用 丝状蛋白Septin 4/5，已被证明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械蛋白。这个 Septins在VSMCMV分泌中的作用尚不清楚，我们对PLCγ2/Septin4/5相互作用的发现以及 来自PLCγ2-/-VSMC的MVS4/5增加支持了Septins在调节VSMC Mv中的新作用 分泌物和钙化。因此，我们推测PLCγ2缺乏可诱导血管平滑肌细胞钙化 通过改变Septin/肌动蛋白-细胞骨架结构，导致MV分泌增加。使用我们的新产品 小鼠模型，目的1确定SMC特异性γ2在体内调节血管功能的功能； 目的2将阐明PLCγ2调控血管平滑肌细胞钙化的分子机制。这些 研究将阐明SMC来源的PLCγ2在调节细胞骨架结构和MV中的综合作用 导致VSMC钙化的分泌物。最近发现人类中的PLCγ2突变会导致 免疫疾病，但其潜在机制尚不完全清楚。因此，描绘出 PLCγ-2调节血管功能的新功能和新机制 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.