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

人类接触环境毒素，如烟草相关产品中的毒素，是导致 美国可预防的死亡，通过推广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 增加冠心病风险还没有被很好地理解。此外，环境中可能参与基因的基因 在人口层面上，相互作用一直很难确定。因此，我们的长期目标是使用相反的方法 研究外源毒素与相关已知CAD相关基因相互作用的遗传学方法。 其中一个基因是芳香烃受体(AHR)。众所周知的AHR的配体是二恶英和多芳基 碳氢化合物是烟草烟雾的主要成分，也是已知的动脉粥样硬化的促进剂 动物模型。编码AHR、其异二聚化伙伴ARNT和该途径中的其他因子的基因 通过人类基因关联研究，都与冠心病风险有关。单细胞RNA测序(ScRNAseq) 对平滑肌细胞(SMC)特异的AhR基因敲除(KO)小鼠的研究表明， 表达软骨细胞标志物的表型转移型SMC比例增加，识别细胞 术语“软骨肌细胞”(CMC)。这些发现与更大的病变范围，更多的血统相关- 可追踪到SMC对斑块的贡献，纤维帽中血统可追溯的SMC减少，病变增加。 Ahr KO小鼠的碱性磷酸酶活力。这些发现表明AHR在SMC中的表达抑制 它们转变为CMC并改善血管疾病的病理生理学。这些数据与 大量研究表明，二恶英等异种配体激活AHR会促进动脉粥样硬化， 并提出了一个独特的假设。我们推测AHR在正常情况下对SMC有有益的影响 疾病背景，抑制有害细胞状态向CMC表型和疾病的转变 这种保护作用被外源毒素激活所阻断。因此，我们建议 通过以下目标来检验这一假设。在目标1中，我们将调查Ahr如何应对 异种生物配体在疾病背景下的激活，与SMC表型和细胞损伤解剖有关。 这些研究将使用Ahr SMC特定的条件性KO和SMC血统追踪的ApoE KO 动脉粥样硬化模型。目标2将集中于异种配体的转录和表观基因组效应。 联合使用scRNAseq和单细胞ATAC，用相同的小鼠疾病模型活体激活 测序(ScATACseq)。最后，在目标3中，我们建议使用人冠状动脉SMC作为体外培养的 用于验证和表征先前目标中确定的TF的下游路径的模型系统 它们与AHR相互作用，调节SMC向软骨肌细胞的表型转变。这些研究 研究一个高度创新的假说，并将提供对细胞和分子的重要见解 烟草和其他环境风险因素促进冠心病风险的机制。

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.