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

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.