KLF4-Dependent Regulation of SMC Differentiation and Phenotypic Switching

描述（由申请人提供）：平滑肌细胞（SMC）分化状态的改变在多种心血管疾病的发生和进展中起关键作用。该项目的长期目标是阐明在血管发育过程中控制SMC生长和分化的细胞和分子机制，以及这些控制过程在与血管损伤和疾病相关的SMC表型转换过程中如何改变。我们最近的研究结果表明，迷人的多能性基因Krupple-Like Factor 4（KLF 4）是培养SMC中SMC表型转换的关键介质，并且在我们实验室产生的条件性KLF 4敲除（KO）小鼠中颈动脉结扎损伤后体内。本提案中的研究将检验以下假设：KLF 4在发育过程中以及在血管损伤或动脉粥样硬化发展后的表型转换过程中，在调节血管SMC分化状态的转变中起关键作用。目的1将确定KLF 4在血管损伤的体内抑制SMC标记基因表达的机制，包括确定KLF 4是否通过以下方式介导抑制SMC基因：a）与大多数SMC启动子中发现的保守G/C抑制子和/或TCE元件结合; B）诱导与转录沉默相关的表观遗传修饰; c）抑制有效的SMC选择性SRF共激活因子myocardin的表达;和/或d）诱导多能性基因。目的2将测试的假设，延迟抑制SMC标记基因，并加剧病变形成后，在我们以前的研究中观察到的条件KLF 4基因敲除小鼠血管损伤，介导的，至少部分是由KLF 4在SMC中的损失，而不是其损失EC或巨噬细胞。研究将包括骨髓移植和SMC特异性条件性KLF 4 KO小鼠损伤研究。目的3是确定条件性或SMC特异性条件性敲除KLF 4是否改变ApoE敲除小鼠动脉粥样硬化内膜病变的大小或细胞组成。目的4是确定KLF 4在激活MMP 3、MCP-1、iNOS和VCAM-1等基因中的作用，这些基因可能在血管损伤或实验性动脉粥样硬化的内膜病变发展和/或进展中发挥重要的功能作用。 公共卫生相关性：已知血管平滑肌细胞（SMC）的分化状态（即细胞特异性性质）的异常控制在许多主要疾病中起关键作用，包括动脉粥样硬化、哮喘、高血压和肿瘤转移。该提案中的研究将为控制SMC在发育和疾病中分化的机制提供新的见解，并可能导致新的更有效的治疗方法。

DESCRIPTION (provided by applicant): Alterations in the differentiated state of the smooth muscle cell (SMC) play a key role in the development and progression of a variety of cardiovascular diseases. The long-term goal of this project is to elucidate cellular and molecular mechanisms that control the growth and differentiation of SMC during vascular development, and how these control processes are altered during phenotypic switching of SMC in association with vascular injury and disease. Results of our recent studies have implicated the fascinating pluripotency gene Krupple-Like Factor 4 (KLF4) as a key mediator of SMC phenotypic switching in cultured SMC, and in vivo following carotid ligation injury in conditional KLF4 knockout (KO) mice generated in our lab. Studies in this proposal will test the hypothesis that KLF4 plays a critical role in regulating transitions in the differentiated state of vascular SMC during development, as well as during phenotypic switching following vascular injury or development of atherosclerosis. Aim 1 will determine mechanisms by which KLF4 represses expression of SMC marker genes in vivo with vascular injury including determining if KLF4 mediates repression SMC genes by: a) binding to conserved G/C repressor and/or TCE elements found in most SMC promoters; b) inducing epigenetic modifications associated with transcriptional silencing; c) suppressing expression of the potent SMC-selective SRF co-activator myocardin; and/or d) inducing pluripotency genes. Aim 2 will test the hypothesis that delayed repression of SMC marker genes and exacerbated lesion formation following vascular injury observed in our previous studies in conditional KLF4 knockout mice, were mediated, at least in part, by loss of KLF4 in SMC rather than its loss in EC or macrophages. Studies will include bone marrow transfer, and SMC specific conditional KLF4 KO studies in mice injury. Aim 3 is to determine if conditional or SMC specific conditional knockout of KLF4 alters atherosclerotic intimal lesion size or cellular composition in ApoE knockout mice. Aim 4 is to determine the role of KLF4 in activation of genes such as MMP3, MCP-1, iNOS, and VCAM-1 which are likely to play an important functional role in intimal lesion development and/or progression in response to vascular injury or experimental atherosclerosis. PUBLIC HEALTH RELEVANCE: Abnormal control of the differentiated state (i.e. cell specific properties) of vascular smooth muscle cells (SMC) is known to play a critical role in a number of major diseases including atherosclerosis, asthma, hypertension, and tumor metastasis. Studies in this proposal will provide novel insights into mechanisms that control SMC differentiation in development and disease and may lead to new and more effective therapies.