Saturday, May 11, 2024
5/11/2024
Smooth muscle cells (SMCs) play a critical role in atherosclerosis and coronary artery disease (CAD) risk. During disease, a small subset of SMCs proliferate extensively, undergo cell fate change(s) and migrate into the lesion in a process termed phenotypic modulation. Using single cell RNA sequencing (scRNAseq), we have shown that this cell state transition is a continuum in which these SMCs first assume a fibroblast-like phenotype and ultimately transition to a calcific phenotype. We have also shown that top CAD-associated genes modify disease risk by altering the process of SMC phenotypic modulation. Our subsequent preliminary scRNAseq data suggest that different modulated SMC phenotypes might arise as the result of discrete cell fate decisions resulting in different transcriptional paths during disease. This suggests that causal CAD genes function by altering the clonal proliferation and/or cell fate determination of SMCs during disease, but these processes cannot be directly observed with traditional scRNAseq approaches. We have recently shown that the transcription factor TWIST1 is the causal gene at 7p22.1, a genomic locus in human GWAS that is associated with multiple vascular diseases including CAD, stroke, peripheral artery disease and Moyamoya disease. Our preliminary data show that SMC-specific Twist1 knockdown: i) decreases lesion size in the ApoE- /- atherosclerotic mouse model, consistent with the directionality predicted by human GWAS, ii) results in a >60% reduction in the number of SMC-derived cells within the lesion and iii) specifically results in depletion of the calcific modulated SMC phenotype in scRNAseq data. In addition, siTwist1 knockdown in human coronary artery SMCs (HCASMCs) reveals a strong reduction in cellular proliferation/viability and migration. Taken together, these data lead to our central hypothesis that the 7p22.1 locus acts through TWIST1 to drive vascular disease risk by promoting SMC proliferation and altering cell fate decisions during phenotypic modulation, which will be explored through three Specific Aims. Aim 1 will use a novel scRNAseq-compatible cell barcoding mouse model to track individual SMCs as they proliferate and undergo cell fate determination during atherosclerosis, with and without Twist1 knockout. In Aim 2, we will use a SMC-specific myc-tagged Twist1 overexpression mouse model to simulate the human risk allele, and determine whether increased Twist1 expression in SMCs is sufficient to worsen atherosclerosis. We will also leverage the expression of myc-Twist1 to query genome-wide Twist1 DNA binding in SMCs, identifying the direct molecular targets of Twist1 in vivo during atherosclerosis. Aim 3 will determine the cellular pathways and transcription factors that act on the causal SNP at 7p21.1 to regulate TWIST1 expression. Completion of these studies will result in a detailed map of the SMC proliferation and lineage determination landscape during atherosclerosis, which will be a major resource for the field. We will determine how Twist1 alters this landscape, and elucidate the detailed molecular mechanisms of how 7p21.1 and TWIST1 affects the risk for multiple vascular diseases in humans.
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