Regulation and Function of Myocardin in Vascular Pathobiology

描述（由申请人提供）：血清反应因子（SRF）和心肌素（心肌素）构成激活血管平滑肌细胞（VSMC）细胞收缩和离子通道基因的分子触发开关，这些基因含有SRF结合的CArG盒。我们首先发现心肌水平（而非SRF）与VSMC分化程度相关。由于VSMC分化程序不是固定的，并且在许多疾病过程中受到心肌水平变化的影响，因此我们对了解心肌表达的调控及其作为维持VSMC静止收缩状态的稳态开关的功能很感兴趣。因此，我们在一系列论文中报道，心肌(a)足以通过srf依赖性的收缩和离子通道基因表达变化赋予VSMC收缩能力；(b)完全抑制骨骼肌分化程序；(c)控制越来越多的microrna，包括microRNA143/145基因；(d) TGF¿1通过p38mapk依赖性途径正向诱导。初步数据进一步证实了转录抑制因子和新的microRNA的顺式元件，以及功能数据，首次显示心肌表达对血管闭塞性疾病、脂质摄取和炎症标志物表达的有利影响。总的来说，我们越来越多的工作为检验心肌是正常VSMC分化的稳态开关这一假设提供了重要的基础。本论文将通过一系列相互关联的特异性目的来验证，旨在阐明心肌表达的转录和转录后控制，以及心肌在损伤诱导的新内膜扩张和动脉粥样硬化过程中血管重构中的作用。在Aim 1中，细菌人工染色体转基因小鼠将阐明导致心肌激活或抑制的新型顺式作用元件的功能。在Aim 2中，新型心肌功能丧失和功能获得小鼠将直接评估这种强大的辅助因子在实验性血管疾病过程中的作用，包括对VSMC炎症、增殖和转分化状态的影响。在Aim 3中，将进行基因组研究，将我们生成的人类和小鼠CArGome数据（bbb84,000 CArG盒）与VSMC的RNA-seq相结合，其中心肌在缺乏或存在SRF的情况下表达。这样的分析将揭示CArGome的一个对心肌反应的亚群，以及在血管疾病中重要的新的不依赖srf的心肌靶基因。因此，计划中的研究将对正常出生后发育和血管疾病过程中心肌表达的体内调控提供新的见解，以及心肌作为与物理损伤和动脉粥样硬化疾病相关的血管重塑的可能抑制剂的新信息。这些研究的结果将在设计新的治疗策略来对抗急性和慢性血管疾病以及其他可能改变心肌表达/活性的疾病（例如哮喘、高血压、阿尔茨海默病）方面具有巨大的应用价值。

DESCRIPTION (provided by applicant): Serum response factor (SRF) and Myocardin (MYOCD) constitute a molecular trigger switch for the activation of a battery of vascular smooth muscle cell (VSMC) cyto-contractile and ion channel genes containing SRF- binding CArG boxes. We first showed that levels of MYOCD (but not SRF) correlate with the degree of VSMC differentiation. Since the VSMC differentiation program is not fixed and subject to adaptation in a number of disease processes where levels of MYOCD change, we have been interested in understanding the regulation of MYOCD expression and its function as a homeostatic switch for the preservation of a quiescent, contractile state in VSMC. Accordingly, we have reported in a series of papers that MYOCD (a) is sufficient for conferring VSMC contractile competence through SRF-dependent changes in contractile and ion channel gene expression; (b) completely represses the program of skeletal muscle differentiation; (c) controls a growing number of microRNAs, including the microRNA143/145 gene; and (d) is positively induced by TGF¿1 through a p38MAPK-dependent pathway. Preliminary data further demonstrate cis elements for a transcriptional repressor and new microRNA as well as functional data showing, for the first time, the favorable effects of MYOCD expression on vascular occlusive disease, lipid uptake, and inflammatory marker expression. Collectively, our growing body of work serves as a critical foundation to test the hypothesis that MYOCD is a homeostatic switch for normal VSMC differentiation. This thesis will be tested through a series of inter-related specific aims designe to elucidate transcriptional and post-transcriptional control of MYOCD expression and the role of MYOCD in vascular remodeling during injury-induced neointimal expansion and atherogenesis. In Aim 1, bacterial artificial chromosome transgenic mice will elucidate the function of novel cis-acting elements leading to activation or repression of MYOCD. In Aim 2, novel MYOCD loss- and gain-of-function mice will directly assess the role of this powerful cofactor in experimental vascular disease processes, including effects on VSMC inflammatory, proliferative, and transdifferentiative states. In Aim 3, genomic studies integrating human and mouse CArGome data we have generated (> 84,000 CArG boxes) with RNA-seq of VSMC where MYOCD is expressed in the absence or presence of SRF will be carried out. Such an analysis will reveal a subset of the CArGome that is responsive to MYOCD as well as new SRF-independent MYOCD target genes of import in vascular disease. Thus, the planned studies will yield new insight into the in vivo regulation of MYOCD expression during normal postnatal development and in vascular disease processes as well as novel information related to MYOCD as a likely inhibitor of vascular remodeling associated with physical injury and atherosclerotic disease. The results of these studies will have enormous applications for devising new therapeutic strategies to combat acute and chronic vascular diseases and perhaps other diseases where MYOCD expression/activity may be altered (e.g., asthma, hypertension, Alzheimer's disease).