P roliferation of vascular smooth muscle cells (VSMCs) is a central axiom of most models of atherosclerosis, promoting atherogenesis as a response to injury 1 or inflammation. 2 However, most heart attacks are caused by rupture of a "vulnerable" plaque with a thin VSMC-poor fibrous cap overlying a relatively large necrotic core. 3,4 Plaque repair requires VSMC proliferation and is thus beneficial at this stage. However, VSMCs from advanced human plaques show poor proliferation and premature senescence in culture 5 and in vivo 6 ; furthermore, fibrous cap VSMCs show extensive DNA damage, marked telomere shortening, and markers of senescence. 7 Although these findings suggest that VSMC senescence may be important in atherogenesis, its mechanisms and direct consequences are unproven. Clinical Perspective on p 1919Replicative cell senescence is mediated in part by telomeres, which shorten during replication and ultimately trigger a DNA damage response (DDR) and growth arrest. Telomeres are composed of tandem DNA repeats that are maintained in a compact T-loop structure by the shelterin complex of telomere-associated proteins, including TRF1, TRF2, POT1, TIN2, RAP1, and TPP1. Shelterin proteins restrict access to telomerase and exonucleases/ligases, thus avoiding inappropriate telomere elongation and shortening, respectively, and prevent exposure of chromosome ends that are recognized as double-stranded DNA breaks (DSBs). Although each of the shelterin proteins is important for telomere maintenance, telomeric repeat-binding factor-2 (TRF2) has a particularly critical role. TRF2 regulates replicative senescence in part by reducing telomere length at senescence 8,9 and can also stop the ataxia telangiectasia kinase (ATM) from initiating a DDR from functional telomeres. 10 Loss of TRF2 induces multiple features of senescence, including irreversible growth arrest, expression of senescence-associated β-galactosidase, and telomere dysfunction with chromosomal fusions.11-13 TRF2 can also regulate cell longevity in a telomere-independent manner by direct association with multiple DDR proteins, including ATM, Nijmegen breakage syndrome-1, and checkpoint kinase-2.14-16 ATM phosphorylates TRF2 after DNA 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 degradat...

Background-Vascular smooth muscle cells (VSMCs) in human atherosclerosis manifest extensive DNA damage and activation of the DNA damage response, a pathway that coordinates cell cycle arrest and DNA repair, or can trigger apoptosis or cell senescence. Sirtuin 1 deacetylase (SIRT1) regulates cell ageing and energy metabolism and regulates the DNA damage response through multiple targets. However, the direct role of SIRT1 in atherosclerosis and how SIRT1 in VSMCs might regulate atherosclerosis are unknown. Methods and Results-SIRT1 expression was reduced in human atherosclerotic plaques and VSMCs both derived from plaques and undergoing replicative senescence. SIRT1 inhibition reduced DNA repair and induced apoptosis, in part, through reduced activation of the repair protein Nijmegen Breakage Syndrome-1 but not p53.

Rationale: DNA damage and the DNA damage response have been identified in human atherosclerosis, including in vascular smooth muscle cells (VSMCs). However, although double-stranded breaks (DSBs) are hypothesized to promote plaque progression and instability, in part, by promoting cell senescence, apoptosis, and inflammation, the direct effects of DSBs in VSMCs seen in atherogenesis are unknown. Objective: To determine the presence and effect of endogenous levels of DSBs in VSMCs on atherosclerosis. Methods and Results: Human atherosclerotic plaque VSMCs showed increased expression of multiple DNA damage response proteins in vitro and in vivo, particularly the MRE11/RAD50/NBS1 complex that senses DSB repair. Oxidative stress–induced DSBs were increased in plaque VSMCs, but DSB repair was maintained. To determine the effect of DSBs on atherosclerosis, we generated 2 novel transgenic mice lines expressing NBS1 or C-terminal deleted NBS1 only in VSMCs, and crossed them with apolipoprotein E −/− mice. SM22α-NBS1/apolipoprotein E −/− VSMCs showed enhanced DSB repair and decreased growth arrest and apoptosis, whereas SM22α-(ΔC)NBS1/apolipoprotein E −/− VSMCs showed reduced DSB repair and increased growth arrest and apoptosis. Accelerating or retarding DSB repair did not affect atherosclerosis extent or composition. However, VSMC DNA damage reduced relative fibrous cap areas, whereas accelerating DSB repair increased cap area and VSMC content. Conclusions: Human atherosclerotic plaque VSMCs show increased DNA damage, including DSBs and DNA damage response activation. VSMC DNA damage has minimal effects on atherogenesis, but alters plaque phenotype inhibiting fibrous cap areas in advanced lesions. Inhibiting DNA damage in atherosclerosis may be a novel target to promote plaque stability.