Smooth Muscle Differentiation and Maturation

描述（申请人提供）：血管平滑肌细胞（SMC）异常分化或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主要心血管疾病的发病和进展中起着至关重要的作用。转化生长因子-2 （TGF-2）及其信号分子在SMC的分化和表型调节中起重要作用。然而，调控TGF-2功能的分子机制，特别是负责SMC分化起始和SMC成熟的下游靶基因，在很大程度上仍然未知。我们发表的数据表明，补体32应答基因（RGC-32），一个新的TGF-2下游靶点，是神经嵴祖细胞向SMC分化的必要条件。初步研究表明，tgf -2诱导的人胚胎干细胞源性间充质干细胞（huMSC）向SMC分化需要RGC-32。此外，RGC-32似乎对SMC分化和成熟都很重要。本研究的目的是验证RGC- 32通过与不同核因子的相互作用激活SMC分化并促进SMC成熟的整体假设。我们提出三个具体的目标来测试这一假设使用分子，细胞和遗传方法的组合。在Aim 1中，我们将验证RGC-32通过与Smad蛋白相互作用激活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Aim 2中，我们将验证RGC-32与心肌素相互作用以刺激SMC成熟的假设。RGC-32在收缩性SMC表型形成中的重要性将通过huMSC和大鼠主动脉SMC的形态学、生化和收缩性实验来确定。RGC-32与心肌素在诱导SMC收缩中的物理和功能相互作用也将被测试。在Aim 3中，我们将验证RGC-32对体内SMC分化至关重要的假设。RGC-32在SMC分化中的作用将通过RGC-32敲除小鼠模型来确定。RGC-32基因敲除对体内SMC标记基因转录激活的影响将采用SM221启动子驱动的LacZ转基因小鼠模型进行测试。总的来说，这些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，这将最终有助于开发治疗或预防SMC相关心血管疾病的新疗法。

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.