Environment-Sensing Aryl Hydrocarbon Receptor Inhibits the Chondrogenic Fate of Modulated Smooth Muscle Cells in Atherosclerotic Lesions

Abstract

Introduction Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. MethodsWe combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease.Results Genomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term "chondromyocytes" (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. ConclusionOverall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.

Smad3Regulates Smooth Muscle Cell Fate and Governs Adverse Remodeling and Calcification of Atherosclerotic Plaque

“…SMC traced cells were identified by expression of the tdTomato gene, and the component of this cluster representing mature medial SMC was identified by expression of SMC lineage markers Myh11 and Cnn1. As we have shown previously, a significant portion of the SMC-lineage traced cells during disease did not express these mature SMC markers and thus represented SMC that had undergone phenotypic transition (24,25,30). We were thus able to determine whether there were alterations in the number of mature differentiated versus transition SMC in Smad3 ΔSMC mice.…”

“…Thus, to better understand how loss of Smad3 expression produced phenotypic changes in lesion SMC derived cells, and their interactions with other lesion cell types, we performed single cell RNA expression profiling (scRNAseq) of atherosclerotic lesions from Smad3 knockout and control animals. The atherosclerotic tissue was harvested, processed for single cell encapsulation, RNA capture, reverse transcription and amplification with the 10X Genomics Chromium V3 platform, and cDNA libraries were sequenced as previously described( 25, 30 ). Subsequently, the single cell expression data were visualized utilizing Uniform Manifold Approximation and Projection (UMAP) to create a 2-D projection representing the organization of individual cells to each other in clusters, and of the relationship of the organized clusters to each other.…”

“…The validity of this clustering approach was further confirmed via visualization of cell population canonical "markers" for all clusters in the UMAP space. Previous work in SMC lineage tracing in atherosclerosis has identified three distinct clusters of SMC derived cells, including mature SMC, fibromyocytes (FMC), and pro-calcific chondromyocytes (CMC) (21,30,35,46). Similar subsets of lineage traced SMC were identified in these data ( To determine if loss of Smad3 expression alters the cell-fate transitions of diseaseassociated SMC lineage cells, we ascertained the fraction of de-differentiated cells that contribute to clusters for each of the two known and the novel transition phenotypes.…”

“…Consistent with this notion, recent lineage tracing studies have demonstrated that the majority of cells inside atherosclerotic plaque, including those expressing some inflammatory markers, are oligoclonal de-differentiated smooth muscle derivatives (26)(27)(28)(29). Genes that alter SMC behavior are known to influence the composition of an atherosclerotic plaque (21,24,25,28,30). CADassociated genes TCF21 and AHR have been linked to these processes, and various genomic data suggests that additional CAD genes might also regulate SMC phenotype (23,(30)(31)(32)(33)(34).…”

“…Genes that alter SMC behavior are known to influence the composition of an atherosclerotic plaque (21,24,25,28,30). CADassociated genes TCF21 and AHR have been linked to these processes, and various genomic data suggests that additional CAD genes might also regulate SMC phenotype (23,(30)(31)(32)(33)(34). While these SMC progenies have been previously lumped together as phenotypically modulated SMC, advances in single cell RNA profiling has demonstrated the presence of subsets of these cells with distinct transcriptomes and cell fates.…”