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

Abstract

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC biology can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC, but the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 promoted SMC progeny to take on a chondromyocyte fate. These analyses also identified a unique subset of smooth muscle lineage cells that promote remodeling and recruitment of inflammatory cells. This population was marked by uniquely high Mmp3 and Cxcl12 expression, and increased in number after loss of Smad3, thus contributing to higher-risk plaque features. Further investigation of transcriptional mechanisms by which Smad3 mediates TGFβ effects on SMC gene expression provided evidence that it binds in conjunction with Hox and Sox transcription factors in the regulatory region of genes that regulate aortic remodeling and have been linked to human Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits cell state changes that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.

Proteoglycan 4 Modulates Osteogenic Smooth Muscle Cell Differentiation during Vascular Remodeling and Intimal Calcification

“…The inhibition of SMC migration and proliferation suggests that PRG4 may both influence calcification and SMC phenotype in atherosclerosis. While changes in SOX9 and SMAD3 levels have already been shown to induce osteogenic SMC transformation [ 64 , 69 , 70 ], our data indicate that, together with a PRG4-enriched matrix, these effects may lead to increased ectopic calcification. Moreover, we speculate that this matrix in turn enables SMCs to restore a gene expression profile resembling a more differentiated phenotype.…”

ZEB2 Shapes the Epigenetic Landscape of Atherosclerosis

“…The SMC phenotype seen here appears to be opposite to the phenotype observed with SMC loss of Smad3 as we have previously observed (ie, increased phenotypic modulation and increased transition to the most fibroblast-like Mmp3 and Cxcl12 expressing remodeling SMC [R-SMC] phenotype cells). 33 With Zeb2 ΔSMC cells, we observed decreased phenotypic modulation as well as decreased expression of characteristic R-SMC genes Mmp3 and Cxcl12 and loss of almost all R-SMC cluster cells (Figure 4K-4N).…”

“…Single-cell RNA sequencing data from control and Zeb2 ΔSMC mice were processed as described previously 13,30,33 and unbiased UMAP clustering identified different cell groups that contribute to the lesion (Figure 4A). As with scATAC-Seq data, loss of Zeb2 resulted in transcriptional alteration and phenotypic change in only the transition SMC population, as marked by lineage tracing (Figure 4A-4C).…”

“…Atherosclerotic lesions in the aortic root were then dissected and processed for scATAC-Seq, scRNA-Seq, or histologic analysis (Figure 3A). 13,17,33 scATAC-Seq data were processed, clustered on the basis of differentially accessible peaks, and visualized using dimensional reduction with UMAP (uniform manifold approximation and projection; Figure 3B). The identity of each cluster was determined using the pseudoexpression metric on the basis of chromatin accessibility around specific marker genes (Figure S3).…”

The role of smooth muscle cells in calcification of atherosclerotic plaques

“…Therefore, these results are a first indication for a more stable expression profile of highly calcified lesions in late-stage atherosclerosis. Gene set enrichment analysis, estimating the impact of dysregulated genes on biological pathways, supported our initial findings (upregulation of SMC differentiation, muscle contraction, ECM organization), but additionally revealed induction of processes involved in both SMC fate and osteogenic transformation, such as SMAD and TGFb signaling 186,330,331 (Figure 9). Repressed biological pathways confirmed mitigation of inflammation, ECM degradation, cholesterol metabolism and cytokine responses, with TNF, IL 1b, IFNg mediated signaling among the most significantly affected.…”