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 are mostly unknown. Similarly, DNA damage and the DNA damage response (DDR) have been identified in human atherosclerosis, including in vascular smooth muscle cells (VSMCs). However, although double strand 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 Methods and Results: We examined expression of proteins that protecting telomeres from shortening 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. DNA damage induced loss of TRF2 by increased ubiquitin-mediated degradation of 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 ApoE-/- mice. TRF2 overexpression bypassed senescence, reduced DNA damage, and accelerated DNA repair, whereas TRF2188A showed opposite effects. Transgenic mice expressing VSMC-specific TRF2T188A showed increased atherosclerosis and necrotic core formation in vivo, whereas VSMC-specific TRF2 increased relative fibrous cap and decreased necrotic core areas. TRF2 protected against atherosclerosis independent of secretion of senescence-associated cytokines. We next examined human plaques for evidence of DNA damage. Human atherosclerotic plaque VSMCs showed increased expression of multiple DDR proteins in vitro and in vivo, particularly the MRN complex (MRE11, RAD50, NBS1) 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 two novel transgenic mice lines expressing NBS1 or C-terminal deleted NBS1 only in VSMCs, and crossed them with ApoE-/- mice. SM22a-NBS1/ApoE-/- VSMCs showed enhanced DSB repair and decreased growth arrest and apoptosis, whereas SM22a-(DC)NBS1/ApoE-/- 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: We conclude that plaque VSMC senescence in atherosclerosis is associated with loss of TRF2. VSMC senescence promotes both atherosclerosis and features of plaque vulnerability, identifying prevention of senescence as a potential target for intervention. Human atherosclerotic plaque VSMCs also show increased DNA damage including DSBs and DDR 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 also be a novel target to promote plaque stability.