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Aberrant vascular smooth muscle cell (VSMC) homeostasis and proliferation characterize vascular diseases causing heart attack and stroke. Here we elucidate molecular determinants governing VSMC proliferation by reconstructing gene regulatory networks from single-cell transcriptomics and epigenetic profiling. We detect widespread activation of enhancers at disease-relevant loci in proliferation-predisposed VSMCs. We compared gene regulatory network rewiring between injury-responsive and nonresponsive VSMCs, which suggested shared transcription factors but differing target loci between VSMC states. Through in silico perturbation analysis, we identified and prioritized previously unrecognized regulators of proliferation, including RUNX1 and TIMP1. Moreover, we showed that the pioneer transcription factor RUNX1 increased VSMC responsiveness and that TIMP1 feeds back to promote VSMC proliferation through CD74-mediated STAT3 signaling. Both RUNX1 and the TIMP1-CD74 axis were expressed in human VSMCs, showing low levels in normal arteries and increased expression in disease, suggesting clinical relevance and potential as vascular disease targets., (© 2024. The Author(s).)

Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of multiple vascular pathologies, including following neointimal formation after injury and atherosclerosis. However, human VSMCs in advanced atherosclerotic lesions show reduced cell proliferation, extensive and persistent DNA damage, and features of premature cell senescence. Here, we report that stress-induced premature senescence (SIPS) and stable expression of a telomeric repeat-binding factor 2 protein mutant (TRF2 T188A ) induce senescence of human VSMCs, associated with persistent telomeric DNA damage. VSMC senescence is associated with formation of micronuclei, activation of cGAS-STING cytoplasmic sensing, and induction of multiple pro-inflammatory cytokines. VSMC-specific TRF2 T188A expression in a multicolor clonal VSMC-tracking mouse model shows no change in VSMC clonal patches after injury, but an increase in neointima formation, outward remodeling, senescence and immune/inflammatory cell infiltration or retention. We suggest that persistent telomere damage in VSMCs inducing cell senescence has a major role in driving persistent inflammation in vascular disease.

Rationale: Vascular smooth muscle cell (VSMC) senescence promotes atherosclerosis and features of plaque instability, in part, through lipid-mediated oxidative DNA damage and telomere dysfunction. SIRT6 (Sirtuin 6) is a nuclear deacetylase involved in DNA damage response signaling, inflammation, and metabolism; however, its role in regulating VSMC senescence and atherosclerosis is unclear., Objective: We examined SIRT6 expression in human VSMCs, the role, regulation, and downstream pathways activated by SIRT6, and how VSMC SIRT6 regulates atherogenesis., Methods and Results: SIRT6 protein, but not mRNA, expression was markedly reduced in VSMCs in human and mouse atherosclerotic plaques, and in human VSMCs derived from plaques or undergoing replicative or palmitate-induced senescence versus healthy aortic VSMCs. The ubiquitin ligase CHIP (C terminus of HSC70-interacting protein) promoted SIRT6 stability, but CHIP expression was reduced in human and mouse plaque VSMCs and by palmitate in a p38- and c-Jun N-terminal kinase-dependent manner. SIRT6 bound to telomeres, while SIRT6 inhibition using shRNA or a deacetylase-inactive mutant (SIRT6 H133Y ) shortened human VSMC lifespan and induced senescence, associated with telomeric H3K9 (histone H3 lysine 9) hyperacetylation and 53BP1 (p53 binding protein 1) binding, indicative of telomere damage. In contrast, SIRT6 overexpression preserved telomere integrity, delayed cellular senescence, and reduced inflammatory cytokine expression and changes in VSMC metabolism associated with senescence. SIRT6, but not SIRT6 H133Y , promoted proliferation and lifespan of mouse VSMCs, and prevented senescence-associated metabolic changes. ApoE -/- (apolipoprotein E) mice were generated that overexpress SIRT6 or SIRT6 H133Y in VSMCs only. SM22α-hSIRT6/ApoE -/- mice had reduced atherosclerosis, markers of senescence and inflammation compared with littermate controls, while plaques of SM22α-hSIRT6 H133Y /ApoE -/- mice showed increased features of plaque instability., Conclusions: SIRT6 protein expression is reduced in human and mouse plaque VSMCs and is positively regulated by CHIP. SIRT6 regulates telomere maintenance and VSMC lifespan and inhibits atherogenesis, all dependent on its deacetylase activity. Our data show that endogenous SIRT6 deacetylase is an important and unrecognized inhibitor of VSMC senescence and atherosclerosis.

Objective: Vascular inflammation underlies cardiovascular disease. Vascular smooth muscle cells (VSMCs) upregulate selective genes, including MMPs (matrix metalloproteinases) and proinflammatory cytokines upon local inflammation, which directly contribute to vascular disease and adverse clinical outcome. Identification of factors controlling VSMC responses to inflammation is therefore of considerable therapeutic importance. Here, we determine the role of Histone H3 lysine 9 di-methylation (H3K9me2), a repressive epigenetic mark that is reduced in atherosclerotic lesions, in regulating the VSMC inflammatory response. Approach and Results: We used VSMC-lineage tracing to reveal reduced H3K9me2 levels in VSMCs of arteries after injury and in atherosclerotic lesions compared with control vessels. Intriguingly, chromatin immunoprecipitation showed H3K9me2 enrichment at a subset of inflammation-responsive gene promoters, including MMP3 , MMP9 , MMP12 , and IL6 , in mouse and human VSMCs. Inhibition of G9A/GLP (G9A-like protein), the primary enzymes responsible for H3K9me2, significantly potentiated inflammation-induced gene induction in vitro and in vivo without altering NFκB (nuclear factor kappa-light-chain-enhancer of activated B cell) and MAPK (mitogen-activated protein kinase) signaling. Rather, reduced G9A/GLP activity enhanced inflammation-induced binding of transcription factors NFκB-p65 and cJUN to H3K9me2 target gene promoters MMP3 and IL6 . Taken together, these results suggest that promoter-associated H3K9me2 directly attenuates the induction of target genes in response to inflammation in human VSMCs., Conclusions: This study implicates H3K9me2 in regulating the proinflammatory VSMC phenotype. Our findings suggest that reduced H3K9me2 in disease enhance binding of NFκB and AP-1 (activator protein-1) transcription factors at specific inflammation-responsive genes to augment proinflammatory stimuli in VSMC. Therefore, H3K9me2-regulation could be targeted clinically to limit expression of MMPs and IL6, which are induced in vascular disease.

Objective- Recent studies suggested the occurrence of phenotypic switching of vascular smooth muscle cells (VSMCs) during the development of aortic aneurysm (AA). However, lineage-tracing studies are still lacking, and the behavior of VSMCs during the formation of dissecting AA is poorly understood. Approach and Results- We used multicolor lineage tracing of VSMCs to track their fate after injury in murine models of Ang II (angiotensin II)-induced dissecting AA. We also addressed the direct impact of autophagy on the response of VSMCs to AA dissection. Finally, we studied the relevance of these processes to human AAs. Here, we show that a subset of medial VSMCs undergoes clonal expansion and that VSMC outgrowths are observed in the adventitia and borders of the false channel during Ang II-induced development of dissecting AA. The clonally expanded VSMCs undergo phenotypic switching with downregulation of VSMC differentiation markers and upregulation of phagocytic markers, indicative of functional changes. In particular, autophagy and endoplasmic reticulum stress responses are activated in the injured VSMCs. Loss of autophagy in VSMCs through deletion of autophagy protein 5 gene ( Atg5) increases the susceptibility of VSMCs to death, enhances endoplasmic reticulum stress activation, and promotes IRE (inositol-requiring enzyme) 1α-dependent VSMC inflammation. These alterations culminate in increased severity of aortic disease and higher incidence of fatal AA dissection in mice with VSMC-restricted deletion of Atg5. We also report increased expression of autophagy and endoplasmic reticulum stress markers in VSMCs of human dissecting AAs. Conclusions- VSMCs undergo clonal expansion and phenotypic switching in Ang II-induced dissecting AAs in mice. We also identify a critical role for autophagy in regulating VSMC death and endoplasmic reticulum stress-dependent inflammation with important consequences for aortic wall homeostasis and repair.

Background: Atherosclerotic plaques demonstrate extensive accumulation of oxidative DNA damage, predominantly as 8-oxoguanine (8oxoG) lesions. 8oxoG is repaired by base excision repair enzymes; however, the mechanisms regulating 8oxoG accumulation in vascular smooth muscle cells (VSMCs) and its effects on their function and in atherosclerosis are unknown., Methods: We studied levels of 8oxoG and its regulatory enzymes in human atherosclerosis, the mechanisms regulating 8oxoG repair and the base excision repair enzyme 8oxoG DNA glycosylase I (OGG1) in VSMCs in vitro, and the effects of reducing 8oxoG in VSMCs in atherosclerosis in ApoE -/- mice., Results: Human plaque VSMCs showed defective nuclear 8oxoG repair, associated with reduced acetylation of OGG1. OGG1 was a key regulatory enzyme of 8oxoG repair in VSMCs, and its acetylation was crucial to its repair function through regulation of protein stability and expression. p300 and sirtuin 1 were identified as the OGG1 acetyltransferase and deacetylase regulators, respectively, and both proteins interacted with OGG1 and regulated OGG1 acetylation at endogenous levels. However, p300 levels were decreased in human plaque VSMCs and in response to oxidative stress, suggesting that reactive oxygen species-induced regulation of OGG1 acetylation could be caused by reactive oxygen species-induced decrease in p300 expression. We generated mice that express VSMC-restricted OGG1 or an acetylation defective version (SM22α-OGG1 and SM22α-OGG1 K-R mice) and crossed them with ApoE -/- mice. We also studied ApoE -/- mice deficient in OGG1 (OGG1 -/- ). OGG1 -/- mice showed increased 8oxoG in vivo and increased atherosclerosis, whereas mice expressing VSMC-specific OGG1 but not the acetylation mutant OGG1 K-R showed markedly reduced intracellular 8oxoG and reduced atherosclerosis. VSMC OGG1 reduced telomere 8oxoG accumulation, DNA strand breaks, cell death and senescence after oxidant stress, and activation of proinflammatory pathways., Conclusions: We identify defective 8oxoG base excision repair in human atherosclerotic plaque VSMCs, OGG1 as a major 8oxoG repair enzyme in VSMCs, and p300/sirtuin 1 as major regulators of OGG1 through acetylation/deacetylation. Reducing oxidative damage by rescuing OGG1 activity reduces plaque development, indicating the detrimental effects of 8oxoG on VSMC function.

Objective: Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis., Approach and Results: Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E-deficient (ApoE -/- ) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE -/- mice overexpressing the mitochondrial helicase Twinkle (Tw + /ApoE -/- ). Tw + /ApoE -/- mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw + /ApoE -/- mice had decreased necrotic core and increased fibrous cap areas, and Tw + /ApoE -/- bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress-induced apoptosis., Conclusions: Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in reactive oxygen species and may be a promising therapeutic strategy in atherosclerosis., (© 2017 The Authors.)

Background: Although vascular smooth muscle cell (VSMC) proliferation is implicated in atherogenesis, VSMCs in advanced plaques and cultured from plaques show evidence of VSMC senescence and DNA damage. In particular, plaque VSMCs show shortening of telomeres, which can directly induce senescence. Senescence can have multiple effects on plaque development and morphology; however, the consequences of VSMC senescence or the mechanisms underlying VSMC senescence in atherosclerosis are mostly unknown., Methods and Results: We examined the expression of proteins that protect telomeres in VSMCs derived from human plaques and normal vessels. Plaque VSMCs showed reduced expression and telomere binding of telomeric repeat-binding factor-2 (TRF2), associated with increased DNA damage. TRF2 expression was regulated by p53-dependent degradation of the TRF2 protein. To examine the functional consequences of loss of TRF2, we expressed TRF2 or a TRF2 functional mutant (T188A) as either gain- or loss-of-function studies in vitro and in apolipoprotein E(-/-) mice. TRF2 overexpression bypassed senescence, reduced DNA damage, and accelerated DNA repair, whereas TRF2(188A) showed opposite effects. Transgenic mice expressing VSMC-specific TRF2(T188A) showed increased atherosclerosis and necrotic core formation in vivo, whereas VSMC-specific TRF2 increased the relative fibrous cap and decreased necrotic core areas. TRF2 protected against atherosclerosis independent of secretion of senescence-associated cytokines., Conclusions: We conclude that plaque VSMC senescence in atherosclerosis is associated with loss of TRF2. VSMC senes cence promotes both atherosclerosis and features of plaque vulnerability, identifying prevention of senescence as a potential target for intervention., (© 2015 American Heart Association, Inc.)