Receptor-regulated Ca2+ Entry Pathways in Smooth Muscle

平滑肌中受体调节的 Ca2 进入途径

• 批准号: 7793009
• 负责人: Mohamed Trebak
• 金额: $ 39.5万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-19 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7793009
• 关键词: 1,2-diacylglycerol; Adenoviruses; Agonist; Arachidonic Acids; Atherosclerosis; Biochemical; Blood Vessels; Calcium; Calcium Channel; Cell membrane; Cell physiology; Cells; Clinical; Co-Immunoprecipitations; Development; Diglycerides; Disease; Electrophysiology (science); Ensure; Fluorescence Resonance Energy Transfer; Functional disorder; Goals; Growth Factor Receptors; Homologous Gene; Hypertension; Image; Immigration; In Vitro; Injury; Inositol; Ion Channel; Mediating; Microscopy; Modeling; Molecular; Pathway interactions; Pharmacotherapy; Phenotype; Phospholipase C; Platelet-Derived Growth Factor; Play; Proliferating; Proteins; RNA; RNA Interference; Rattus; Regulation; Role; STIM1 gene; Sarcoplasmic Reticulum; Second Messenger Systems; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Testing; Thrombin; Up-Regulation; Vascular Diseases; Vascular Smooth Muscle; angiogenesis; in vivo; injured; insight; leiomyosarcoma; migration; novel; patch clamp; protein expression; public health relevance; receptor; repaired; response; second messenger; sensor; vascular smooth muscle cell proliferation; voltage

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. PUBLIC HEALTH RELEVANCE: 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.

描述（申请人提供）：血管平滑肌细胞（VSMC）增殖和迁移对于血管发育、血管生成和修复至关重要；它们的功能障碍会导致动脉粥样硬化、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物中重现。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疾病中具有至关重要的临床重要性，但这种调节的分子机制仍不完全清楚。 VSMC 表型调节伴随着离子通道表达的变化：合成 VSMC 下调 L 型 Ca2+ 通道的表达并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，与静态的新鲜分离细胞相比，合成培养的大鼠主动脉 VSMC 中 TRPC6 和新发现的钙传感器 STIM1 以及钙通道 Orai1 和 Orai3 的表达有所增加。我们假设 STIM1 是 Orai1、Orai3 和 TRPC6 通道功能所需的 VSMC 中 Ca2+ 信号传导的主要调节因子，并且 STIM1、Orai 和 TRPC 上调导致的 Ca2+ 进入增加有助于疾病中的 VSMC 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 可抑制培养物中的 VSMC 增殖，并且我们揭示了不同 Ca2+ 通道的激动剂特异性激活以及 STIM1 在调节这些通道方面的显着多功能性。事实上，在合成 VSMC 中敲低 STIM1 会消除以下功能： i) PDGF 激活的 Orai1 通道； ii) 由异多聚体 Orai1/3 贡献的凝血酶激活通道和 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 相互作用在此 Ca2+ 进入途径激活中的作用。在 Aim2 中，我们将确定肌浆网或质膜相关的 STIM1 是否参与 DAG 的 TRPC6 激活，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，并使用免疫共沉淀检查其天然对应物的相互作用。在 Aim3 中，我们将测试以下假设：Orai1、Orai3 和 TRPC6 上调也发生在大鼠血管损伤模型的体内，并确定使用编码 siRNA 的腺病毒体内沉默这些蛋白质对新内膜形成的影响。该提案的结果将有助于更好地了解 VSMC 生理学，并揭示旨在控制动脉粥样硬化和高血压等血管疾病期间发生的 VSMC 增殖和迁移的药物治疗新靶点。 公共健康相关性：该提案的结果将有助于更好地了解 VSMC 生理学，并揭示旨在控制动脉粥样硬化和高血压等血管疾病期间发生的 VSMC 增殖和迁移的药物治疗新靶点。