Smooth Muscle Differentiation and Maturation

描述(由申请人提供)：众所周知，血管平滑肌细胞(SMC)分化异常或SMC表型改变在动脉粥样硬化、先天性心脏病、主动脉瘤、高血压和再狭窄等几种突出的心血管疾病状态的发生和发展中起着关键作用。转化生长因子-2(TGF-2)及其信号分子在SMC分化和表型调控中起重要作用。然而，调控转化生长因子-2功能的分子机制，特别是参与SMC分化和成熟的下游靶基因仍然很大程度上是未知的。我们已发表的数据表明，补体32反应基因(RGC-32)是一种新的转化生长因子-2下游靶点，对于SMC从神经脊祖细胞分化是必不可少的。初步研究表明，RGC-32是转化生长因子-2诱导人胚胎干细胞来源的间充质干细胞向SMC分化所必需的。此外，RGC-32似乎对SMC的分化和成熟都很重要。本提案的目的是验证RGC-32激活SMC分化并通过与不同核因子相互作用促进SMC成熟的总体假设。我们提出了三个具体的目标，使用分子、细胞和遗传方法的组合来检验这一假设。在目标1中，我们将验证RGC-32通过与Smad蛋白相互作用而激活SMC分化的假设。通过检测RGC-32与Smad蛋白在激活早期SMC标记基因1-SMA和SM221转录途径中的物理和功能相互作用，探讨RGC-32在SMC分化中的作用机制。在目标2中，我们将验证RGC-32与myocardin相互作用以刺激SMC成熟的假设。RGC-32在人MSC和大鼠主动脉SMC形成收缩SMC表型中的重要性将通过形态、生化和收缩能力的测定来确定。还将测试RGC-32与myocardin在诱导SMC收缩方面的物理和功能相互作用。在目标3中，我们将验证RGC-32在体内对SMC分化是必不可少的假设。RGC-32基因敲除小鼠模型将确定RGC-32在SMC分化中的作用。利用SM221启动子驱动的LacZ转基因小鼠模型，检测RGC-32基因敲除对体内SMC标志基因转录激活的影响。总而言之，计划中的研究将对控制SMC分化和成熟的细胞/分子机制产生新的见解，这最终将有助于开发治疗或预防SMC相关心血管疾病的新疗法。 公共卫生相关性：血管平滑肌细胞分化是血管发育过程中的一个非常重要的过程，众所周知，这个过程中的变化在几种主要心血管疾病的发生和发展中发挥了作用，包括动脉粥样硬化、先天性心脏病、主动脉瘤、高血压和再狭窄。我们的建议是研究调节平滑肌分化的分子机制，这将有助于确定这些疾病的治疗靶点。

DESCRIPTION (provided by applicant): It is well recognized that abnormal vascular smooth muscle cell (SMC) differentiation or alterations in SMC phenotype play critical roles in the pathogenesis and progression of several prominent cardiovascular disease states including atherosclerosis, congenital heart diseases, aortic aneurysm, hypertension, and restenosis. Transforming growth factor-2 (TGF-2) and its signaling molecules play important roles in SMC differentiation and phenotypic modulation. The molecular mechanisms governing TGF-2 function, especially the downstream target genes responsible for the initiation of SMC differentiation and SMC maturation, however, remains largely unknown. Our published data have shown that response gene to complement 32 (RGC-32), a novel TGF-2 downstream target, is essential for SMC differentiation from neural crest progenitor cells. Preliminary studies demonstrate that RGC-32 is required for TGF-2-induced SMC differentiation from human embryonic stem cell- derived mesenchymal stem cells (huMSC). Moreover, RGC-32 appears to be important for both SMC differentiation and maturation. The goals of the current proposal are to test the overall hypothesis that RGC- 32 activates SMC differentiation and promote SMC maturation through interaction with different nuclear factors. We propose three specific aims to test this hypothesis using a combination of molecular, cellular and genetic approaches. In Aim 1, we will test the hypothesis that RGC-32 activates SMC differentiation by interacting with Smad proteins. The mechanism underlying RGC-32 function in SMC differentiation will be explored by testing the physical and functional interaction of RGC-32 with Smad proteins in activating the transcription pathway of early SMC marker genes 1-SMA and SM221. In Aim 2, we will test the hypothesis that RGC-32 interacts with myocardin to stimulate SMC maturation. The importance of RGC-32 in the formation of contractile SMC phenotype will be determined by morphological, biochemical and contractility assay in huMSC and rat aorta SMC. The physical and functional interaction of RGC-32 with myocardin in inducing SMC contraction will also be tested. In Aim 3, we will test the hypothesis that RGC-32 is essential for SMC differentiation in vivo. The role of RGC-32 in SMC differentiation will be determined using RGC-32 knockout mouse model. The effect of RGC-32 gene knockout on the activation of SMC marker gene transcription in vivo will be tested using SM221 promoter-driven LacZ transgenic mouse model. Collectively, the planned studies will yield novel insight into cellular/molecular mechanisms that control SMC differentiation and maturation, which will ultimately contribute to the development of novel therapeutics for the treatment or prevention of SMC-related cardiovascular diseases. PUBLIC HEALTH RELEVANCE: Vascular smooth muscle cell differentiation is a very important process during the development of blood vessels and it is well recognized that alterations in this process play a role in the pathogenesis and progression of several prominent cardiovascular disease states including atherosclerosis, congenital heart diseases, aortic aneurysm, hypertension, and restenosis. Our proposal examining the molecular mechanisms that regulate smooth muscle differentiation should help to identify therapeutic targets for the treatment of these diseases.