Identifying tobacco-genetic interactions through study of the aryl hydrocarbon receptor pathway.

人类暴露于环境毒素，如烟草相关产品中的毒素，是导致 在美国，可预防的死亡，通过宣传对发病率和死亡率的影响最大 冠状动脉疾病（CAD）。然而，环境暴露的分子机制 增加CAD风险还没有被很好地理解。此外，可能通过环境参与基因的基因 在人口水平上很难确定相互作用。因此，我们的长期目标是使用反向 遗传学方法来研究外源毒素与相关已知CAD相关基因的相互作用。 一个这样的基因是芳基烃受体（AHR）。众所周知的AHR配体是二恶英和聚芳基 碳氢化合物是烟草烟雾的主要成分，也是动脉粥样硬化的已知促进剂， 动物模型编码AHR、其异源二聚化伴侣ARNT和该途径中的其他因子的基因 都与CAD风险通过人类遗传关联研究。单细胞RNA测序（scRNAseq） 对平滑肌细胞（SMC）特异性Ahr敲除（KO）动脉粥样硬化小鼠的研究显示， 表达软骨细胞标志物的表型转变SMC比例增加， 术语“软骨肌细胞”（CMC）。这些发现与较大的病变大小，增加的谱系- 追踪SMC对斑块的贡献，减少纤维帽中的谱系追踪SMC，增加病变 Ahr KO小鼠中的碱性磷酸酶活性。这些结果表明，平滑肌细胞中Ahr的表达抑制了 它们向CMC的转变并改善血管疾病的病理生理学。这些数据与A 许多研究表明，异生物质配体如二恶英激活Ahr会促进动脉粥样硬化， 提出了一个独特的假设我们假设，Ahr通常对SMC有有益的影响， 疾病背景，抑制有害细胞状态向CMC表型的转变，以及疾病 这种保护作用被外源性毒素激活所阻断。因此，我们建议， 通过以下目的来检验这个假设。在目标1中，我们将研究Ahr如何响应 外源性配体激活的疾病设置，相对于SMC表型和细胞病变解剖。 这些研究将采用Ahr SMC特异性条件性KO和SMC谱系示踪ApoE KO 动脉粥样硬化模型目的2将集中在转录组和表观基因组效应的外源性配体， 用相同的小鼠疾病模型，用组合的scRNAseq和单细胞ATAC进行体内活化 测序（scATACseq）。最后，在目标3中，我们提出采用人冠状动脉SMC作为体外培养的血管平滑肌细胞。 模型系统，以验证和表征之前目标中确定的TF的下游途径 与AHR相互作用，调节SMC向软骨肌细胞的表型转变。这些研究 调查一个高度创新的假设，并将提供重要的见解，细胞和分子 烟草和其他环境风险因素促进CAD风险的机制。

Human exposure to environmental toxins such as those in tobacco related products are the leading cause of preventable deaths in the United States, with the greatest effect on morbidity and mortality through promotion of coronary artery disease (CAD). However, the molecular mechanisms by which environmental exposures increase CAD risk are not well understood. Furthermore, genes that might participate in gene by environment interactions have been difficult to identify at the population level. Thus, our longterm goal is to use a reverse genetics approach to study the interaction of xenobiotic toxins with relevant known CAD-associated genes. One such gene is the aryl-hydrocarbon receptor (AHR). Well-known ligands of AHR are dioxins and poly-aryl hydrocarbons, which are major components of tobacco smoke and known promoters of atherosclerosis in animal models. Genes encoding AHR, its heterodimerization partner ARNT, and other factors in this pathway are all linked to CAD risk through human genetic association studies. Single cell RNA sequencing (scRNAseq) studies of smooth muscle cell (SMC)-specific Ahr knockout (KO) atherosclerotic mice showed a significant increase in the proportion of phenotypic transition SMC that express chondrocyte markers, identifying cells we term “chondromyocytes” (CMC). These findings were correlated with larger lesion size, increased lineage- traced SMC contribution to the plaque, decreased lineage-traced SMC in the fibrous cap, and increased lesion alkaline phosphatase activity in the Ahr KO mice. These findings reveal that Ahr expression in SMC inhibits their transition to CMC and ameliorates vascular disease pathophysiology. These data are in contrast with a number of studies showing that Ahr activation by xenobiotic ligands such as dioxin promote atherosclerosis, and suggest a unique hypothesis. We postulate that Ahr normally has a beneficial effect on SMC in the disease setting, inhibiting a harmful cell state transition to the CMC phenotype and disease progression, and that this protective effect is blocked by xenobiotic toxin activation. We thus propose to examine this hypothesis through the following Aims. In Aim 1, we will investigate how Ahr responds to xenobiotic ligand activation in the disease setting, with respect to SMC phenotype and cellular lesion anatomy. These studies will employ the Ahr SMC-specific conditional KO and SMC lineage traced ApoE KO atherosclerosis model. Aim 2 will focus on the transcriptomic and epigenomic effects of xenobiotic ligand in vivo activation with the same mouse disease model, with combined scRNAseq and single cell ATAC sequencing (scATACseq). Finally, in Aim 3 we propose to employ human coronary artery SMC as an in vitro model system to validate, and characterize the downstream pathways for, TFs identified in the previous Aim that interact with AHR to regulate the phenotypic transition of SMC to chondromyocytes. These studies investigate a highly innovative hypothesis, and will provide significant insights into cellular and molecular mechanisms by which tobacco and other environmental risk factors promote CAD risk.