The SMAD3 signaling network in coronary artery disease risk

项目摘要/摘要 转化生长因子β信号通路在血管疾病中已被广泛研究，但仍存在 关于这条途径如何影响作用的方向和机制，存在着相当大的争议 人类冠心病(CAD)。最近的全基因组关联研究已经确定了几个 含有转化生长因子β家族基因的基因座，包括编码转录的15q22.33处的smad3基因。 将转化生长因子β诱导的胞质信号转化为基因表达变化的关键因子和ZEB2 和SKI分别位于2q22.3和1p36.33，与Smad3结合并调节其转录活性。 该实验室的研究采用了组蛋白修饰、染色体可及性、等位基因特异性 表达、体外基因组编辑和转基因报告鼠研究确定Smad3是病因 基因位于15q22.33。该基因的保护性等位基因破坏了Smad3中一个有效的内含子增强子。 与血管组织中SMAD3表达降低相关的基因，提示 SMC中SMAD3的存在增加了CAD的风险。在平滑肌细胞中，转化生长因子β信号被认为具有重要的作用。 分化和抗增殖在血管发育中的作用，但这种作用可能是有害的 在疾病环境中，SMC去分化和增殖，“表型调节”，允许这一点 支持结构完整性和延缓斑块破裂的细胞类型。转化生长因子β的促病作用是 由我们对TCF21的研究支持，TCF21是一种促进SMC表型的CAD相关转录因子 其保护性等位基因可增加其表达。总而言之，这些数据表明 我们的中心假设：转化生长因子β信号分子Smad3与ZEB2和SKI一起， 调节转录网络，该网络介导适应性SMC表型反应 血管应激，等位基因变异调节这一反应，从而导致冠心病风险。 在ApoE-/-动脉粥样硬化小鼠模型中，目标1提出的实验将评估疾病解剖学 SMC特有的Smad3缺失，以及基因组创造的人类风险和保护性单倍型 正在编辑。谱系追踪和单细胞rna-seq将定义Smad3在调节细胞 对疾病刺激的反应，以及作为病变SMC去分化的分子表型，有助于 巨噬细胞谱系。在目标2中，人类冠状动脉SMC的CHIP-SEQ和RNA-SEQ研究将定义 Smad3调控的基因网络，并研究ZEB2和SKI如何表达 调节这个网络的分子组成。AIM人冠状动脉SMC的体外研究 3将确定SMAD3介导的细胞和分子过程以及这些过程是如何发挥作用的 被ZEB2和SKI修改。综上所述，这些研究将极大地促进我们对 Smad3及其相关因子ZEB2和SKI如何调控SMC对血管的表型反应 疾病，以及它们的功能紊乱如何导致冠心病风险。

PROJECT SUMMARY/ABSTRACT The TGFβ signaling pathway has been extensively studied in vascular disease, but there remains considerable controversy regarding the direction and mechanism of effect for how this pathway impacts human coronary artery disease (CAD). Recent genome-wide association studies have identified several loci that harbor TGFβ family genes, including the SMAD3 gene at 15q22.33 that encodes a transcription factor critical for converting TGFβ-induced cytoplasmic signaling to gene expression changes, and ZEB2 and SKI at 2q22.3 and 1p36.33 respectively, which bind SMAD3 and modulate its transcriptional activity. Studies in this lab have employed histone modification, chromosomal accessibility, allele-specific expression, in vitro genome editing and transgenic reporter mouse studies to identify SMAD3 as the causal gene at 15q22.33. The protective allele for this gene disrupts a potent intronic enhancer in the SMAD3 gene that is associated with decreased SMAD3 expression in vascular tissues, suggesting that expression of SMAD3 in SMC promotes risk for CAD. In SMC, TGFβ signaling is known to have important differentiative and anti-proliferative roles during vascular development, but this function may be deleterious in the disease setting where SMC dedifferentiation and proliferation, “phenotypic modulation,” allows this cell type to bolster structural integrity and retard plaque rupture. A disease-promoting role for TGFβ is supported by our studies with TCF21, a CAD associated transcription factor that promotes SMC phenotypic modulation and whose protective allele confers increased expression. Taken together, these data suggest our Central Hypothesis: the TGFβ signaling molecule SMAD3, in conjunction with ZEB2 and SKI, regulates a transcriptional network that mediates the adaptive SMC phenotypic response to vascular stress, with allelic variation modulating this response contributing to CAD risk. Experiments proposed in Aim 1 in the ApoE-/- atherosclerosis mouse model will evaluate disease anatomy with SMC-specific deletion of Smad3, along with human risk and protective haplotypes created by genome editing. Lineage tracing and single cell RNA-seq will define the role of Smad3 in regulating the cellular response to disease stimuli, and molecular phenotype as lesion SMC dedifferentiate and contribute to the macrophage lineage. In Aim 2, ChIP-seq and RNA-seq studies in human coronary artery SMC will define the network of genes that are regulated by SMAD3 and investigate how expression of ZEB2 and SKI modulates the molecular composition of this network. In vitro studies in human coronary artery SMC in Aim 3 will identify the cellular and molecular processes that are mediated by SMAD3 and how these functions are modified by ZEB2 and SKI. Taken together, these studies will significantly advance our understanding of how SMAD3 and related factors ZEB2 and SKI govern the SMC phenotypic response to vascular disease, and how perturbation of their function contributes to CAD risk.