S-EPMC8066499 - Environment-Sensing Aryl Hydrocarbon Receptor Inhibits the Chondrogenic Fate of Modulated Smooth Muscle Cells in Atherosclerotic Lesions. - OmicsDI

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

ABSTRACT:

Background

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 risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC.

Methods

We combined RNA-sequencing, chromatin immunoprecipitation followed by sequencing, assay for transposase-accessible chromatin using sequencing, and in vitro assays in human coronary artery SMCs, with single-cell RNA-sequencing, 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 human coronary artery SMCs revealed that AHR modulates the human coronary artery SMC 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 SMCs in the atherosclerotic lesion cap. Furthermore, single-cell RNA-sequencing studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMCs expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term "chondromyocytes," 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 SMCs in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout in comparison with wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses.

Conclusions

Overall, we conclude that AHR promotes the maintenance of lesion cap integrity and diminishes the disease-related SMC-to-chondromyocyte transition in atherosclerotic tissues.

SUBMITTER: Kim JB

PROVIDER: S-EPMC8066499 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aryl-hydrocarbon receptor protects against endochondral ossification of modulated smooth muscle cells in atherosclerosis 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. Methods: We 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. Conclusion: Overall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.

Project description:Genome-wide association studies (GWAS) for coronary artery disease (CAD, also termed coronary heart disease, CHD) have discovered and validated 48 loci genome-wide and recent 1000-Genomes based meta-analyses have discovered additional 8 loci with significant association, however, true follow-up studies on the mechanisms of association are still scarce. Here we analyze the relationship of two transcription factors, TCF21, one of the lead GWAS candidate genes for coronary artery disease and AHR, aryl hydrocarbon receptor, which previously was not implicated as fundamental for the atherosclerotic process. We show that both the predicted and in-vivo ChIP-Seq binding sites for TCF and AHR colocalize in the human genome with over 400 predicted sites and 119 ChIP-Seq sites directly overlapping. TCF and AHR-ARNT predicted binding sites longitudinal phasing was observed near the transcription start sites, outlining the position of +1 nucleosome. AHR-ARNT matrix was highly enriched in both TCF21 ChIP-Seq peaks and ATAC-Seq open chromatin regions in human coronary artery smooth muscle cells (HCASMC), implicating the role of AHR in this important cell type for vascular biology. Co-expression modules of TCF21 and AHR showed high degree of connectivity. Separation of rotationally phased and unphased predicted binding sites for TCF and AHR resolved the roles of direct and indirect TCF-AHR interactions, and implicated as highly-modulated various inflammatory, interleukin and cytokine related processes, while ChIP-Seq co-localization of TCF21 and AHR/ARNT emphasized the role of calcium related processes. We performed TCF21 overexpression analysis in human coronary artery smooth muscle cells (HCASMC) and obtained GO ontologies that recapitulate in vivo pathophysiology of the atherosclerotic vessel wall, and a set of chronic inflammatory ontologies was established with the colocalization of TCF21 and AHR ChIP-Seq sites as well as with the binomial testing of GWAS SNP overrepresentation. We experimentally confirmed that TCF21 binds to and regulates AHR gene expression in HCASMC, as well as to elements near AHR downstream genes, such as CYP1A1, using reporter assays. The functional relevance of AHR pathway in HCASMC is confirmed using AHR ligands TCDD and oxidized LDL. Finally, we show that AHR is elevated in atherosclerotic arteries using laser capture microdissection in mice in vivo and in human ex vivo. In conclusion, we extend GWAS results to functional assays and show that TCF21 and AHR functional connectivity provides a novel mechanism for diseased coronary vessel wall biology.