A transcriptional network which governs smooth muscle transition is mediated by causal coronary artery disease gene PDGFD

项目摘要或摘要 目前已发现160多个与冠状动脉疾病(CAD)相关的遗传位点， 然而，除了降脂疗法外，这些全基因组关联研究(GWAS)的前景是 识别致病基因和产生新的治疗机制尚未完成。这是迫切需要的 确定致病基因并表征细胞信号、转录和表观基因组调控 发现治疗冠心病的新方法。我导师的实验室已经确定TCF21是CAD 相关基因由GWA6q23.2定位。单细胞转录分析与疾病病变细胞 解剖学研究表明，TCF21在SMC中上调，以促进去分化、增殖和 内侧SMC迁移到斑块中，在那里它们有助于保护纤维帽。我们已经开始 血小板源CAD相关基因对TCF21表达的上游调控 生长因子D(PDGFD)。PDGFD是受体酪氨酸激酶配体家族的成员之一，该家族一直很好地 在SMC表型和动脉粥样硬化的背景下进行研究，并与冠心病有遗传关联 人类。导致PDGFD表达增加的基因变异与恶化的冠心病相关。这些 这些发现使我们把注意力集中在它在CAD中的决定作用上。重要的是，PDGFD还直接 调节其他有效的CAD基因，包括CDKN2B、LMOD1、CXCL12、SMAD3和Twist1，因此 提示它在冠心病风险的调节中起着基础性作用。我们假设PDGFD促进 它通过调节TCF21和其他关键的疾病相关转录因子来调节CAD风险 SMC对血管应激的表型反应。为了解决这一假设，我们将使用一种新的Pdgfd-/-x ApoE- /-结合最先进的平滑肌肉特定报告背景的小鼠模型 同时评估单细胞基因转录和染色质可及性的技术 在血管组织中结合scRNAseq和scATACseq。我们将用两个例子来说明我们的主要假设 主要的具体目标，1)确定Pdgfd在调节SMC表型和细胞和分子中的作用 用小鼠体内模型研究动脉粥样硬化血管组织的特征，以及2)绘制 与疾病相关的转录程序和因果表观遗传调控特征 Pdgfd在动脉粥样硬化小鼠基因敲除模型中的细胞和分子效应。本研究 将使我们对PDGFD如何影响SMC表型反应从而影响CAD有一个基本的了解 通过调节染色质可及性和转录因子结合而产生的风险。完成本建议书 将解决基本问题：(1)Pdgfd表达的扰动如何影响SMC的表型 体内动脉粥样硬化血管组织的分子特征？(2)Pdgfd如何激活表观遗传学和 介导SMC表型调节从而导致冠心病风险的转录过程？此外，这是一个至关重要的 了解PDGFD作为治疗冠心病的潜在治疗靶点的步骤。

PROJECT SUMMARY OR ABSTRACT There are more than 160 genetic loci discovered which are associated with coronary artery disease (CAD), yet, outside of lipid lowering therapies, the promise of these genome wide association studies (GWAS) to identify causal genes and result in novel mechanisms for treatment has not been fulfilled. It is of critical need to identify causal genes and characterize the cellular signaling, transcriptomic, and epigenomic regulation of disease to discover novel treatments of CAD. The lab of my mentor has identified TCF21 as the CAD associated gene mapped by GWAS at 6q23.2. Single cell transcriptomic analysis and disease lesion cellular anatomy studies show that TCF21 is upregulated in SMC to promote de-differentiation, proliferation, and migration of medial SMC into the plaque where they contribute to the protective fibrous cap. We have begun to study the upstream regulation of TCF21 expression by the CAD associated gene encoding platelet derived growth factor D (PDGFD). PDGFD is a member of a receptor tyrosine kinase ligand family that has been well studied in the context of SMC phenotype and atherosclerosis and has a genetic association with CAD in humans. Gene variants resulting in increased PDGFD expression are associated with worsened CAD. These findings have led us to focus our attention on its determinative role in CAD. Importantly, PDGFD also directly regulates other validated CAD genes, including CDKN2B, LMOD1, CXCL12, SMAD3, and TWIST1, thus suggesting that it has a fundamental role in the modulation of CAD risk. We hypothesize that PDGFD promotes CAD risk through its regulation of TCF21 and other key disease related transcription factors that mediate the SMC phenotypic response to vascular stress. To address this hypothesis, we will use a novel Pdgfd-/- x ApoE- /- mouse model on a smooth muscle specific reporter background in combination with state-of-the-art technology to simultaneously evaluate single cell gene transcription and chromatin accessibility through combination scRNAseq with scATACseq in vascular tissue. We will address our primary hypothesis with two major specific aims, 1) Define the role of Pdgfd in regulating SMC phenotype and cellular and molecular features of atherosclerotic vascular tissues with an in-vivo mouse model, and 2) Map the transcriptomic program and causal epigenetic regulatory features that mediate the disease related cellular and molecular effects of Pdgfd in-vivo in the atherosclerosis mouse knockout model. This study will give us a fundamental understanding of how PDGFD influences SMC phenotypic response and thus CAD risk through modulation of chromatin accessibility and transcription factor binding. Completion of this proposal will address fundamental questions: (1) How does perturbation of Pdgfd expression affect the SMC phenotype and molecular features of atherosclerotic vascular tissue in vivo? (2) How does Pdgfd activate epigenetic and transcriptional processes which mediate SMC phenotypic modulation and thus CAD risk? Further, it is a crucial step in understanding PDGFD as a potential therapeutic target in the treatment of CAD.