Premature cell senescence promotes vascular smooth muscle cell phenotypic modulation and resistance to re-differentiation

Abstract

Aims: Human atherosclerotic plaque cells display DNA damage that if left unrepaired can promote premature cell senescence. Vascular smooth muscle cells (VSMCs) predisposed to senescence promote atherogenesis and features of unstable plaques and increase neointima formation after injury. However, how premature VSMC senescence promotes vascular disease and its effects on VSMC phenotype are unknown. Methods and Results: Bulk RNA-seq of primary human VSMCs identified 126 significantly up- or down-regulated genes after both DNA damage-induced (D+R) or replicative senescence (RS). Upregulated genes included senescence markers CDKN2A (p16) and ICAM1 and genes expressed by phenotypically modulated de-differentiated/’fibromyocytic’ VSMCs (osteoprotegerin (TNFRSF11B), fibromodulin (FMOD)) as well as transmembrane protein 178B (TMEM178B) and secreted frizzle-related protein 4 (SFRP4). Mouse VSMCs also upregulated genes associated with de-differentiated VSMC phenotype, Tmem178b and Sfrp4 after D+R. Single-cell RNA-sequencing of lineage-traced VSMCs in mouse plaques or human plaques showed that VSMCs expressing Cdkn2a had lower contractile marker expression and higher expression of de-differentiated VSMC markers. Mice expressing a VSMC-restricted mutant telomere protein (TRF2T188A) that induces premature senescence showed increased atherosclerosis, expression of multiple de-differentiation genes in plaques and after injury, and differential regulation of pathways associated with extracellular matrix organisation, inflammation and Transforming Growth Factor- (Tgfb). Trf2T188A VSMCs were more resistant to re-differentiation and had dysregulated Tgfb signalling at multiple levels with downregulated ligand, receptors, and coactivators and upregulated co-repressor expression. Trf2T188A VSMCs also showed cytosolic DNA and activation of the STING-TBK1-IRF3 pathway that suppressed Tgfb signalling. Silencing IRF3 restored expression of Tgfb pathway components and VSMC contractile markers after TGFb administration. Conclusions: DNA damage and senescence induce genes associated with de-differentiated/fibromyocytic VSMCs, and persistence of these cells in vivo. Failure of senescent VSMCs to re-express contractile markers during re-differentiation suggests that VSMC senescence may promote atherosclerosis and neointima formation in part by inhibiting their re-differentiation.