Atheroprotective roles of smooth muscle cell phenotypic modulation and the TCF21 disease gene as revealed by single-cell analysis

Abstract

AUTHOR CONTRIBUTIONS R.C.W. designed and performed all scRNAseq experiments, analyzed the scRNAseq data, performed the RNAscope in-situ hybridization assays, performed and analyzed the CITE-seq and FACS experiments, analyzed the immunofluorescence data, performed the eQTL analyses, assisted with mouse colony breeding, drafted the manuscript, and led the study. D.W. assisted with the design of the scRNAseq experiments and performed scRNAseq capture and library preparation for all samples. D.T.P. performed scRNAseq capture and helped obtain human coronary samples. J.C. assisted with the scRNAseq capture, library preparation and sequencing. T.N. performed qPCR experiments, analyzed the qPCR data and performed TCF21 ChIPseq. M.P., C.L.M., B.L. and S.B.M. performed the eQTL analyses. R.K. performed the immunohistochemistry experiments and bred the mouse colonies. M.N. performed and analyzed immunohistochemistry experiments. K.Z., M.A. and R.C. assisted with network analysis. T.K.K., R.F. and Y.J.W. prepared the human tissue samples. M.D.T. and J.C.W. provided critical expert guidance on the manuscript. J.B.K. helped plan the mouse in situ histology studies, managed the mouse colonies, performed the TCF21 over-expression experiment and performed the quantitative immunohistochemistry analysis of lesion characteristics. T.Q. conceived and supervised the study. All authors discussed the results and contributed critical review to the manuscript.

LncRNA de novo discovery reveals noncoding RNAs as major molecular mechanism associating coronary artery disease GWAS variants with causal genes to confer disease risk

“…Once HCASMC reach the top of the lipid core they start secreting extracellular matrix proteins and create a firm barrier called fibrous cap that blocks the lipid core from expansion and interaction with the blood flow. Even though modulated SMCs previously have been described as a cell type that contributes to the disease progression, we have shown in our recent work that phenotypic modulation is clearly a beneficial mechanism that protects from lesion rupture, hence HCASMC migrating and contributing to the lesion represent a specific defense mechanism that organism triggers upon lipid injury (Wirka et al 2019). Using lineage tracing in mice with a permanent fluorophore to label the cells that expressed Tcf21 at the time of recombinase construct activation, we have previously shown that TCF21 expressing cells in lesions give rise to smooth muscle cells in the fibrous cap (Nurnberg et al 2015).…”

“…TCF21 expression levels were also highly correlated with the phenotypic modulation as measured with single cell RNASeq in coronary arteries in mice with induced disease progression (high fat diet) and in atherosclerotic human coronary arteries (Wirka et al 2019). TCF21 weighted gene expression score marked a cluster of modulated SMCs in tSNE clustering of single cell RNASeq (Wirka et al 2019). In addition, human genetics showed the association of increased expression of TCF21 in HCASMC with decreased CAD disease risk, indicating protective roles for both TCF21 and phenotypic modulation (Wirka et al 2019).…”

“…We have previously shown that TCF21 represents a potent factor in HCASMC that can shape the future of this cell type during embryogenesis and athero-development (Kim et al 2017;Nurnberg et al 2015;Sazonova et al 2015;Wirka et al 2019). One of the most important protective mechanisms during lesion formation and expansion is the phenotypic modulation of HCASMC.…”

“…The migration of TCF21 cells from the media to through the lesion toward the fibrous cap was spotted using Tcf21-LacZ functional knockout and Xgal staining in mice with disease progression (8 week high fat diet) (Nurnberg et al 2015). TCF21 expression levels were also highly correlated with the phenotypic modulation as measured with single cell RNASeq in coronary arteries in mice with induced disease progression (high fat diet) and in atherosclerotic human coronary arteries (Wirka et al 2019). TCF21 weighted gene expression score marked a cluster of modulated SMCs in tSNE clustering of single cell RNASeq (Wirka et al 2019).…”

“…HCASMC have been previously shown to be crucial in regulation of CAD disease progression, and it is estimated that the phenotypic modulation of HCASMC, i.e. their de-differentiation and migration towards the top of the growing lipid core and formation of the firm barrier, named fibrous cap, represent a protective mechanism engaged by the organism fighting the lipid injury (Wirka et al 2019). We performed deep sequencing of 48 HCASMC samples divided into 6 experimental conditions (pro-differentiation conditions: TGFbeta, TCF21KD; de-differentiation conditions: TNFalpha, PDGFD, Serum, SMAD3KD) and 2 controls (CtrlCyto, treated only with BSA as a control for cytokine treatments and CtrlRNA containing mock transfection used as a control for transfection treatments) and obtained on average 300M reads per sample.…”