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

PDGFD 调节转录网络以调节平滑肌细胞转变和冠状动脉疾病风险

• 批准号: 10385753
• 负责人: THOMAS QUERTERMOUS
• 金额: $ 67.51万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10385753
• 关键词: Alleles; Anatomy; Animals; Apolipoprotein E; Aryl Hydrocarbon Receptor; Atherosclerosis; Autocrine Communication; Binding; Blood Vessels; CRISPR interference; CXCL12 gene; Cardiovascular Diseases; Cell Lineage; Cell model; Cells; Chromatin; Complement Factor D; Coronary Arteriosclerosis; Coronary artery; Coupled; Data; Development; Disease; Enhancers; Epigenetic Process; Fibroblasts; Functional disorder; Gene Expression; Gene Targeting; Genes; Genetic Risk; Genetic Transcription; Goals; Human; Human Genome; In Vitro; Knock-out; Knockout Mice; LacZ Genes; Link; MADH3 gene; Maps; Measures; Mediating; Modeling; Molecular; Morphology; Mus; PDGFA gene; PDGFRB gene; Phenotype; Platelet-Derived Growth Factor; Probability; Process; Regulation; Reporter; Research; Risk Assessment; Role; Scientist; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Subcellular Anatomy; TWIST1 gene; Therapeutic; Tissues; Transcription Process; Transcriptional Activation; Wild Type Mouse; Work; XCL1 gene; cell type; disorder risk; gene function; genome wide association study; genome-wide; in vivo; mouse model; programs; public health relevance; receptor; response; single-cell RNA sequencing; transcription factor; transcriptome; transcriptomics; vascular stress; whole genome

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.

我们已经确定TCF 21为冠状动脉疾病（CAD）相关基因，通过全基因组定位， 在6q23.2的关联研究，并采用了许多机制的方法来表明，它促进了一个 平滑肌细胞（SMC）向成纤维细胞样“纤维肌细胞”表型的转变，以及这些细胞的贡献。 细胞到保护性纤维帽。我们对另一个CAD相关基因--芳香烃的研究 受体（AHR）的作用，其特征在于SMC向第二个软骨形成的“软骨肌细胞”的转变 表型为了扩展这项工作并研究TCF 21上游的表观遗传信号传导机制， AHR和其他介导SMC细胞状态的因子，我们将重点放在CAD相关的血小板 衍生生长因子D基因（PDGFD）。我们已经表明，PDGFD调节TCF 21和其他经验证的 CAD基因包括LMOD 1、CXCL 12和SMAD 3，主要在疾病过渡期SMC中表达， 也表达PDGFRB受体。总之，这些数据表明，PDGFD激活了自分泌 调节SMC表型和CAD风险的信号通路。指导这项研究的假设 假设PDGFD通过调节TCF 21和其他关键疾病促进CAD风险 相关转录因子介导SMC对血管应激的表型反应。主 因此，本文提出的工作目标是鉴定PDGFD靶转录因子（TF）， SMC转换和表征其在这种细胞类型中的转录程序。具体而言，在目标1中，我们将 在ApoE敲除小鼠动脉粥样硬化模型中使用Pdgfd敲除和SMC谱系追踪来表征 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。 形态学和细胞解剖学。在目标2中，我们将在Pdgfd中进行单细胞RNA测序（scRNAseq） 空和野生型动脉粥样硬化小鼠，以表征Pdgfd下游SMC基因表达程序 in this cell细胞type类型.在相同动物中的单细胞ATAC测序（scATACseq）将绘制增强子基因组- 广泛的差异调节SMC表型转换，并确定特定的TF结合这些 增强子以调节纤维肌细胞和软骨肌细胞特异性基因的表达。在目标3中，我们 在PDGFD刺激的体外干扰促进目标2中鉴定的TF的候选SMC转变 人冠状动脉平滑肌细胞去分化模型，以及所得的转录组和细胞 在这个模型的PDGFD功能的上下文中解释的状态效应。这些研究将PDGFD与CAD联系起来 我们在SMC表型转变的背景下表征的相关基因（TCF 21，AHR， SMAD 3、TWIST 1），以及调节SMC表型的另外的高概率CAD基因，以扩增SMC表型。 疾病的转录网络在这种血管细胞类型。这项工作将促进我们对 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， CAD风险。