PDGFD regulates a transcriptional network to modulate smooth muscle cell transition and coronary artery disease risk

我们已经确定TCF21是由全基因组定位的冠心病相关基因 协会在6q23.2进行的研究，并使用了许多机械论方法来表明它促进了 血管平滑肌细胞(SMC)向成纤维细胞样表型的转变及其作用 细胞连接到保护性纤维帽。我们对另一种CAD相关基因--芳香烃的研究 受体(AHR)，表征了SMC向第二个软骨性“软骨肌细胞”的转变。 表型。为了扩展这项工作并研究TCF21上游的表观遗传信号机制， AHR和其他调节SMC细胞状态的因素，我们正专注于CAD相关的血小板 衍生生长因子D基因(PDGFD)。我们已经证明了PDGFD调节TCF21和其他有效的 CAD基因包括LMOD1、CXCL12和SMAD3，主要在疾病转移性SMC中表达 也表达PDGFRb受体。总之，这些数据表明，PDGFD激活了一种自分泌 调节SMC表型和冠心病风险的信号通路。指导这项研究的假设 假设PDGFD通过其对TCF21和其他关键疾病的调节来增加冠心病风险 介导SMC对血管应激的表型反应的相关转录因子。初级阶段 因此，这里提出的工作的目标是识别PDGFD靶标转录因子(TF)， SMC的转变及其在这种细胞类型中的转录程序的特征。具体地说，在目标1中，我们将 利用Pdgfd基因敲除和SMC系谱示踪在ApoE基因缺失的小鼠动脉粥样硬化模型中的特征 该基因对SMC细胞状态转换的影响，以及干扰这些转换对疾病的影响 形态和细胞解剖学。在目标2中，我们将在Pdgfd中进行单细胞RNA测序(scRNAseq 空白和野生型动脉粥样硬化小鼠Pdgfd下游SMC基因表达程序的特征 在这种细胞类型中。在相同动物中的单细胞ATAC测序(ScATACseq)将映射增强子基因组- 在SMC表型转换中受到差异调控的广泛基因，并识别与这些基因结合的特定转录因子 调节纤维肌细胞和软骨肌细胞特异性基因表达的增强剂。在《目标3》中，我们将 在体外刺激的PDGFD中，扰动候选SMC转变促进AIM 2中鉴定的TF 人冠状动脉平滑肌细胞去分化模型及由此产生的转录和细胞 在该模型中，状态效应在PDGFD函数的背景下被解释。这些研究将把PDGFD与CAD联系起来 我们在SMC表型转变(TCF21，AHR， Smad3，Twist1)，以及额外的调节SMC表型的高概率CAD基因，以扩大 这种血管细胞类型中的疾病转录网络。这项工作将增进我们对 动脉粥样硬化的病理生理学和促进靶向血管壁分子过程的努力以改善 CAD风险。

We have identified TCF21 as the coronary artery disease (CAD) associated gene mapped by genome-wide association studies at 6q23.2 and employed numerous mechanistic approaches to show that it promotes a smooth muscle cell (SMC) transition to a fibroblast like “fibromyocyte” phenotype, and the contribution of these cells to the protective fibrous cap. Our studies with another CAD associated gene, the aryl hydrocarbon receptor (AHR), have characterized the transition of SMC to a second, chondrogenic “chondromyocyte” phenotype. To extend this work and investigate the mechanisms of epigenetic signaling upstream of TCF21, AHR, and other factors that mediate SMC cell state, we are focusing efforts on the CAD associated platelet derived growth factor D gene (PDGFD). We have shown that PDGFD regulates TCF21 and other validated CAD genes including LMOD1, CXCL12, and SMAD3, and is expressed primarily in disease transition SMC that also express the PDGFRB receptor. Together, these data suggest that PDGFD activates an autocrine signaling pathway that modulates SMC phenotype and CAD risk. The hypothesis directing this research postulates 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. The primary goals of the work proposed here are thus to identify the PDGFD target transcription factors (TFs) that regulate SMC transitions and characterize their transcriptional program in this cell type. Specifically, in Aim 1 we will employ Pdgfd knockout and SMC lineage tracing in the ApoE null mouse atherosclerosis model to characterize the effect of this gene on SMC cell state transitions, and the impact of perturbing these transitions on disease morphology and cellular anatomy. In Aim 2, we will conduct single cell RNA sequencing (scRNAseq) in Pdgfd null and wildtype atherosclerotic mice to characterize the SMC gene expression program downstream of Pdgfd in this cell type. Single cell ATAC sequencing (scATACseq) in the same animals will map enhancers genome- wide that are differentially regulated in SMC phenotypic transitions, and identify specific TFs that bind these enhancers to regulate expression of fibromyocyte and chondromyocyte specific genes. In Aim 3, we will perturb candidate SMC transition promoting TFs that are identified in Aim 2, in vitro in a PDGFD stimulated human coronary artery smooth muscle cell de-differentiation model, and the resulting transcriptomic and cell state effects interpreted in the context of PDGFD function in this model. These studies will link PDGFD to CAD associated genes that we have characterized in the context of SMC phenotypic transition (TCF21, AHR, SMAD3, TWIST1), and to additional high probability CAD genes that regulate SMC phenotype, to expand the disease transcriptional network in this vascular cell type. This work will advance our understanding of atherosclerosis pathophysiology and promote efforts to target vascular wall molecular processes to ameliorate CAD risk.