Receptor-regulated Ca2+ Entry Pathways in Smooth Muscle

描述（申请人提供）：血管平滑肌细胞（VSMC）的增殖和迁移对血管发育、血管生成和修复至关重要；它们的功能障碍导致动脉粥样硬化、高血压微血管重塑和平滑肌肉瘤等血管疾病。在这些疾病中，VSMC从静止分化和收缩表型转变为合成增殖和迁移表型，这种情况可以在培养中重现。有明确的证据表明，这种VSMC表型调节在动脉粥样硬化和其他血管闭塞性疾病中具有重要的临床意义，但这种调节的分子机制仍不完全清楚。VSMC的表型调节伴随着离子通道表达的变化：合成VSMC下调l型Ca2+通道的表达，上调典型瞬时受体电位（TRPC）通道的表达。我们的初步研究表明，与新鲜分离的静止细胞相比，人工培养的大鼠主动脉VSMC中TRPC6和新发现的钙传感器STIM1以及钙通道Orai1和Orai3的表达增加。我们假设STIM1是VSMC中Orai1、Orai3和TRPC6通道功能所需的Ca2+信号的主要调节因子，并且STIM1、Orai和TRPC上调导致Ca2+进入增加，有助于疾病中VSMC的增殖和迁移。为了支持这一假设，我们发现使用沉默RNA （siRNA）敲低STIM1抑制培养中的VSMC增殖，我们发现激动剂特异性激活不同的Ca2+通道和显著的STIM1多功能性调节这些通道。实际上，合成VSMC中STIM1的敲低取消了以下功能：i) pdgf激活的Orai1通道；ii)异聚物Orai1/3和iii)二酰基甘油（DAG）激活的TRPC6通道。在Aim1中，我们将使用全细胞膜片钳对凝血酶激活的Ca2+进入途径进行生物物理表征，并确定STIM1寡聚化、细胞定位以及与Orai1和Orai3的相互作用在激活Ca2+进入途径中的作用。在ai2中，我们将确定肌浆网或质膜相关的STIM1是否参与DAG激活TRPC6，通过FRET显微镜检查STIM1寡聚化的作用以及STIM1和TRPC6的相互作用，并使用共免疫沉淀检查它们的天然对偶物的相互作用。在aims中，我们将在血管损伤大鼠模型中验证Orai1， Orai3和TRPC6上调也发生在体内的假设，并确定使用腺病毒编码siRNA在体内沉默这些蛋白对新内膜形成的影响。这一研究结果将有助于我们更好地理解VSMC的生理机制，并揭示新的药物治疗靶点，以控制血管粥样硬化和高血压等血管疾病中VSMC的增殖和迁移。

DESCRIPTION (provided by applicant): Vascular smooth muscle cell (VSMC) proliferation and migration are essential for vascular development, angiogenesis and repair; their dysfunction contributes to vascular diseases such as atherosclerosis, hypertensive microvessel remodeling and Leiomyosarcomas. In these diseases VSMC switch from a quiescent differentiated and contractile phenotype to a synthetic proliferative and migratory phenotype, a condition that can be recapitulated in culture. There is clear evidence that this VSMC phenotypic modulation is of paramount clinical importance in atherosclerosis and other vascular occlusive diseases, yet the molecular mechanisms of this modulation remain incompletely understood. VSMC phenotypic modulation is accompanied by a change in ion channel expression: synthetic VSMC downregulate the expression of L-type Ca2+ channels and upregulate that of canonical transient receptor potential (TRPC) channels. Our preliminary studies have demonstrated an increase in TRPC6 and the newly discovered calcium sensor STIM1, and calcium channels Orai1 and Orai3 expression in synthetic cultured rat aortic VSMC, as compared to quiescent freshly isolated cells. We hypothesize that STIM1 is a master regulator of Ca2+ signaling in VSMC required for Orai1, Orai3 and TRPC6 channel function and that increased Ca2+ entry as a result of STIM1, Orai and TRPC upregulation contribute to VSMC proliferation and migration in disease. In support of this hypothesis we found that STIM1 knockdown using silencing RNA (siRNA) inhibited VSMC proliferation in culture and we revealed agonist-specific activation of distinct Ca2+ channels and remarkable STIM1 versatility in regulating these channels. Indeed, knockdown of STIM1 in synthetic VSMC abrogated the function of: i) PDGF-activated Orai1 channels; ii) thrombin-activated channels contributed by heteromultimeric Orai1/3 and iii) Diacylglycerol (DAG)-activated TRPC6 channels. In Aim1 we will biophysically characterize thrombin-activated Ca2+ entry pathways using whole cell patch clamp and determine the role of STIM1 oligomerization, cellular localization and interaction with Orai1 and Orai3 in the activation of this Ca2+ entry pathway. In Aim2 we will determine whether sarcoplasmic reticulum- or plasma membrane- associated STIM1 is involved in TRPC6 activation by DAG, examine the role of STIM1 oligomerization and the interaction of STIM1 and TRPC6 by FRET microscopy and the interaction of their native counterparts using co-immunoprecipitations. In Aim3 we will test the hypothesis that Orai1, Orai3 and TRPC6 upregulation also occurs in vivo in a rat model of vascular injury and determine the effect on neointimal formation of in vivo silencing of these proteins using adenovirus encoding siRNA. The results from this proposal will generate a better understanding of VSMC physiology and unveil novel targets for drug therapy aimed at controlling VSMC proliferation and migration that occur during vascular diseases such as atherosclerosis and hypertension.