Molecular mechanisms of vascular calcification and their connection to coronary disease risk

谱系追踪和单细胞RNA测序(ScRNAseq)相结合在小鼠动脉粥样硬化中的应用 模型在我们对血管疾病的理解中产生了范式转变，表明病变是平滑的 肌肉细胞(SMC)经历表型转化为具有多种复杂表型的衍生细胞。我们 通过全基因组关联定位TCF21为冠心病相关基因 研究表明，该基因调控疾病相关的SMC向成纤维细胞样细胞的转化 表型，产生我们称之为“纤维肌细胞”的细胞。此外，我们和其他人已经证明，内侧SMC可以 也过渡到第二种SMC衍生的细胞表型，其特征是已知的 它们在软骨内骨形成中的作用，充实和扩展了先前研究这一问题的工作 与血管内膜钙化有关的过程。我们证明了这个软骨形成过程，这给了我们 我们称之为“软骨肌细胞”(chondromycell，CMC)的细胞，受两个CAD相关基因的抑制，一个是 编码TGFB1信号分子SMAD3，另一个编码环境敏感芳基 烃受体(AHR)。在小鼠模型中，这两个基因的敲除(KO)显示增加了向 CMC，病变范围较大，血管钙化增多。这些研究确定SOX9是主要驱动因素 表型向CMC表型的转变。我们的长期目标是阐明分子 调节有害的CMC过渡的机制。我们的中心假设是SOX9是 在血管壁和软骨内骨中是这一软骨形成过程的关键启动者 它在SMC中的形成、表达和功能的调节与血管密切相关 钙化和疾病风险。因此，我们的目标是确定上游表观遗传信号 调节SOX9的表达，以及SOX9的表达如何促进CMC的发育和血管 钙化。具体地说，在目标1中，我们将在ApoE KO小鼠中使用Sox9 KO和SMC血统追踪 动脉粥样硬化模型，以表征该基因对SMC细胞状态转变的影响，以及 扰乱了疾病形态和细胞解剖上的这些转变。在目标2中，我们将进行scRNAseq 在这些小鼠中，为了表征SMC基因的表达程序，Sox9下游的这种细胞类型。单人 转座酶可及染色质测序(ScATACseq)在相同动物中的细胞分析将映射 基因组范围内在CMC表型转换中差异调节的增强子，并识别特定的 转录因子结合这些增强子来调节CMC基因的表达。建议进行的研究 在目标3中，将使用体外研究来表征转录和表观遗传机制，通过 SOX9与抑制因子SMAD3和AHR以及促进向CMC转化的新型转录因子相互作用 表型。拟议的研究将确定介导SMC转变的细胞和分子机制 CMC，以及这一过程与血管钙化和疾病风险的关系。

The combination of lineage tracing and single cell RNA sequencing (scRNAseq) in mouse atherosclerosis models has created a paradigm shift in our understanding of vascular disease, showing that lesion smooth muscle cells (SMC) undergo phenotypic transitions into derivative cells with multiple complex phenotypes. We identified TCF21 as a coronary artery disease (CAD) associated gene mapped by genome-wide association studies (GWAS) and showed that this gene regulates a disease-related transition of SMC to a fibroblast like phenotype, producing cells we term “fibromyocytes.” Further, we and others have shown that medial SMC can also transition to a second SMC-derived cellular phenotype, characterized by expression of genes known for their role in endochondral bone formation, substantiating and expanding previous work investigating this process that is linked to intimal vascular calcification. We showed that this chondrogenic process, which gives rise to cells we term “chondromyocytes” (CMC), is actively inhibited by two CAD associated genes, one encoding the TGFB1 signaling molecule SMAD3, and the other encoding the environmental sensing aryl hydrocarbon receptor (AHR). Knockout (KO) of both genes in mouse models showed increased transition to CMC, larger lesion size and increased vascular calcification. These studies identified SOX9 as a primary driver of the phenotypic transition to the CMC phenotype. Our longterm goal is to elucidate the molecular mechanisms that mediate the detrimental CMC transition. Our Central Hypothesis postulates that SOX9 is a key initiator of this chondrogenic process in the vascular wall, as it is in endochondral bone formation, and regulation of its expression and function in SMC is intimately linked to vascular calcification and disease risk. Our objective is thus to determine the upstream epigenetic signals that modulate SOX9 expression, and how SOX9 expression contributes to CMC development and vascular calcification. Specifically, in Aim 1 we will employ Sox9 KO and SMC lineage tracing in the ApoE KO 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 scRNAseq in these mice to characterize the SMC gene expression program downstream of Sox9 in this cell type. Single cell assay of transposase accessible chromatin sequencing (scATACseq) in the same animals will map enhancers genome-wide that are differentially regulated in CMC phenotypic transition, and identify specific transcription factors (TFs) that bind these enhancers to regulate expression of CMC genes. Studies proposed in Aim 3 will employ in vitro studies to characterize the transcriptional and epigenetic mechanism by which SOX9 interacts with the inhibitory factors SMAD3 and AHR, and novel TFs that promote transition to the CMC phenotype. The proposed studies will identify cellular and molecular mechanisms that mediate SMC transition to CMC, and the relationship of this process to vascular calcification and disease risk.