PPARG-dependent Mechanisms Control Endothelial-Smooth Muscle Coordination, Arterial Pressure, Vasomotor Function and Arterial Stiffness

总结/摘要 血管在动脉血压的调节中起着重要的作用。精确BP调节 需要在内皮细胞（EC）中血管舒张和血管收缩信号之间的协调， 肌肉（SMC）。EC衍生的一氧化氮（NO）是指示SMC扩张或收缩的关键信号之一。 合同我们的研究表明，NO通路是通过转录和后表达协同调控的。 核受体转录因子PPARγ启动的翻译途径。我们的数据支持这一 概念认为，PPARγ：1）在EC中作为传感器调节氧化还原状态，并通过此调节NO的生物利用度， 和2）通过独立控制a）RhoA/Rho激酶， （ROCK）促进收缩的活性，和B）环GMP（cGMP）的产生和稳定性，环GMP是一种关键的 血管舒张介质。在两种细胞类型中，PPARγ依赖性分子机制的范围是 令人惊讶地复杂;需要新的转录辅因子（例如视黄醇结合蛋白7; RBP 7），其形成 一个转录调控中心与过氧化物酶体增殖物激活受体γ，和翻译后调控的关键SMC介质（RhoA 和磷酸二酯酶5，PDE 5）通过Cullin-3E 3泛素连接酶介导的蛋白质周转。重要的是这 PPARγ启动的“最终共同通路”对血管功能、血压和血管内皮细胞的功能有着重要的影响。 僵硬，并且本文提出的研究对这些疾病的治疗具有潜在的意义。 然而，启动和介导这些反应的信号和分子靶点的范围仍然存在， 不太了解。该提案将重点关注两种不同的PPARγ调节途径。我们会研究 PPARγ-RhoBTB 1-Cullin-3通路，并将1）确定RhoBTB 1-Cullin-3通路是否 可以通过评估RhoBTB 1是否可以保护和逆转 表型在高血压模型或其他疾病模型中的应用，其中血管功能障碍是高血压的主要原因。 同时，2）确定RhoBTB 1在包括内皮在内的其他细胞类型中是否重要，以及3）采用 蛋白质组学策略鉴定血管平滑肌中新的RhoBTB 1结合伴侣和Cullin-3底物 肌肉.我们还将检查内皮中的PPARγ-RBP 7-抗氧化途径，并将进行1） 结构功能分析，以确定RBP 7调节PPARγ转录活性的关键机制，以及 2)全基因组转录组和染色质免疫沉淀研究，以评估RBP 7的贡献 对PPARγ及其异源二聚体RXR介导的转录活性的影响。该计划将导致新的 的概念和方向的调查领域，不仅将加深我们的理解的作用， 血管系统中的两个根本性的重要途径，但也将解决基本的转录和 在许多细胞类型中具有相关性的翻译后机制。

Summary/Abstract Blood vessels play an important role in the regulation of arterial blood pressure (BP). Precise BP regulation requires coordination between vasodilator and vasoconstrictor signals in the endothelium (EC) and smooth muscle (SMC). EC-derived nitric oxide (NO) is among the key signals which instruct the SMC to dilate or contract. Our studies show that the NO pathway is coordinately regulated through transcriptional and post- translational pathways initiated by PPARγ, a nuclear receptor transcription factor. Our data support the concepts that PPARγ: 1) acts as a sensor in EC to regulate redox state, and through this, bioavailability of NO, and 2) regulates the responsiveness of SMC to NO by independently controlling a) a RhoA/Rho kinase (ROCK) activity that promotes constriction, and b) production and stability of cyclic GMP (cGMP), a critical mediator of vasodilation. The range of PPARγ-dependent molecular mechanisms in both cell types is surprisingly complex; requiring novel transcriptional co-factors (e.g. retinol binding protein 7; RBP7) which form a transcriptional regulatory hub with PPARγ, and post-translational regulation of critical SMC mediators (RhoA and phosphodiesterase 5, PDE5) by Cullin-3 E3 ubiquitin ligase-mediated protein turnover. Importantly, this PPARγ initiated “final common pathway” has profound effects on vasomotor function, BP and vascular stiffness, and the studies proposed herein have potential implications for the treatment of these disorders. However, the signals which initiate and mediate these responses and the range of molecular targets remain poorly understood. This proposal will focus on two distinct PPARγ-regulated pathways. We will examine the PPARγ-RhoBTB1-Cullin-3 pathway in smooth muscle and will 1) determine if the RhoBTB1-Cullin-3 pathway can be exploited as a potential future therapeutic by assessing if RhoBTB1 can protect and reverse phenotypes in models of hypertension or in other disease models in which vascular dysfunction is a comorbidity, 2) determine if RhoBTB1 is important in other cells types including endothelium, and 3) employ a proteomic strategy to identify novel RhoBTB1 binding partners and Cullin-3 substrates in vascular smooth muscle. We will also examine the PPARγ-RBP7-anti-oxidant pathway in endothelium and will perform 1) structure function analysis to identify key mechanisms regulating PPARγ transcriptional activity by RBP7, and 2) genome wide transcriptome and chromatin immunoprecipitation studies to assess the contribution of RBP7 to mediated transcriptional activity of PPARγ and its obligate heterodimer RXR. This program will lead to new concepts and directions of investigation for the field and will not only deepen our understanding of the role of two fundamentally important pathways in the vasculature, but will also address fundamental transcriptional and post-translational mechanisms that are of relevance in many cell types.