Rationale: DNA damage is present in both genomic and mitochondrial DNA in atherosclerosis. However, whether DNA damage itself promotes atherosclerosis, or is simply a byproduct of the risk factors that promote atherosclerosis, is unknown. Objective:To examine the effect of DNA damage on atherosclerosis, we studied apolipoprotein (Apo)E ؊/؊ mice that were haploinsufficient for the protein kinase ATM (ataxia telangiectasia mutated), which coordinates DNA repair. Methods and Results: ATM

Abstract-Although monocytes/macrophages are considered important in atherogenesis, their role in established plaques is unclear. For example, macrophage content is associated with plaque instability, but their loss through cell death is observed at sites of plaque rupture. To examine the role of monocytes/macrophages in atherosclerosis, we developed CD11b-diphtheria toxin (DT) receptor (DTR) transgenic mice, whereby administration of DT selectively kills monocytes/macrophages. DT treatment reduced peripheral blood monocytes and tissue macrophages and inhibited macrophage function in CD11b-DTR mice and apolipoprotein E-null (apoE Ϫ/Ϫ ) mice transplanted with CD11b-DTR bone marrow. In atherogenesis experiments, DT markedly reduced plaque development and altered plaque composition, reducing collagen content and necrotic core formation. In mice with established plaques, acute DT treatment induced macrophage apoptosis and reduced macrophage content but did not induce plaque inflammation, thrombosis, or rupture. Furthermore, despite a 50% reduction in monocytes, chronic DT treatment of these mice did not alter plaque extent or composition, most likely because of ongoing recruitment/proliferation of monocytes with recovery of macrophage content. We conclude that monocytes/macrophages are critical to atherogenesis, but established plaques are more resistant to reductions in monocytes. (Circ Res. 2007;100:884-893.)

Background— Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species. Methods and Results— mtDNA damage occurred early in the vessel wall in apolipoprotein E–null (ApoE −/− ) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE −/− mice deficient for mitochondrial polymerase-γ proofreading activity (polG −/− /ApoE −/− ). polG −/− /ApoE −/− mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG −/− /ApoE −/− mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG −/− /ApoE −/− bone marrow increased the features of plaque vulnerability, and polG −/− /ApoE −/− monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden. Conclusions— We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.

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.

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Abstract-Although the hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used in atherosclerosis to reduce serum cholesterol, statins have multiple other effects, including direct effects on cells of the vessel wall. Recently, DNA damage, including telomere shortening, has been identified in vascular smooth muscle cells (VSMCs) in human atherosclerosis. Although statins reduce DNA damage in vitro, the mechanisms by which they might protect DNA integrity in VSMCs are unknown. We show that human atherosclerotic plaque VSMCs exhibit increased levels of double-stranded DNA breaks and basal activation of DNA repair pathways involving ataxia telangiectasia-mutated (ATM) and the histone H2AX in vivo and in vitro. Oxidant stress induced DNA damage and activated DNA repair pathways in VSMCs. Statin treatment did not reduce oxidant stress or DNA damage but markedly accelerated DNA repair. Accelerated DNA repair required both the Nijmegen breakage syndrome (NBS)-1 protein and the human double minute protein Hdm2, accompanied by phosphorylation of Hdm2, dissociation of NBS-1 and Hdm2, inhibition of NBS-1 degradation, and accelerated phosphorylation of ATM. Statin treatment reduced VSMC senescence and telomere attrition in culture, accelerated DNA repair and reduced apoptosis in vivo after irradiation, and reduced ATM/ATR (ATM and Rad3-related) activity in atherosclerosis. We conclude that statins activate a novel mechanism of accelerating DNA repair, dependent on NBS-1 stabilization and Hdm2. Statin treatment may delay cell senescence and promote DNA repair in atherosclerosis. H uman atherosclerotic plaques demonstrate evidence of DNA damage, including expression of oxidized guanosine residues, DNA strand breaks, and activation of DNA repair enzymes. 1,2 Although DNA damage is seen in both vascular smooth muscle cells (VSMCs) and macrophages, the mechanisms underlying DNA damage and its biological consequences are unknown. For example, DNA damage can promote apoptosis and premature cell senescence (reviewed elsewhere 3 ), both of which are prominent in VSMCs in human atherosclerosis. 2,4 Conversely, accelerating DNA repair may prevent or reduce accumulated DNA damage, preventing apoptosis or cell senescence.DNA damage induces a cascade of activated proteins that act as sensors and effectors of the damage response, to stall the cell cycle allowing repair to occur, to promote repair, or to induce apoptosis if damage is severe. DNA damage activates Nijmegen breakage syndrome (NBS)-1, a ubiquitously expressed 754-aa protein and key regulator of the MRE11/RAD-50/NBS-1 (MRN) complex. 5,6 MRN promotes early processing of double-strand breaks (DSBs) via DNA binding and nuclease activities, functions as a DSB sensor, and also recruits the ataxia telangiectasia-mutated (ATM) protein to DSBs, followed by ATM activation. 7-9 ATM is normally present as inactive dimers, but DSB exposure induces autophosphorylation at Ser1981, dimer dissociation, and kinase activation. ATM has multiple downstream substrates that me...

Abstract-Recent studies have indicated that the tumor suppressor gene p53 limits atherosclerosis in animal models; p53 expression is also increased in advanced human plaques compared with normal vessels, where it may induce growth arrest and apoptosis. However, controversy exists as to the role of endogenous levels of p53 in different cell types that comprise plaques. We examined atherosclerotic plaque development and composition in brachiocephalic arteries and aortas of p53 Ϫ/Ϫ /ApoE Ϫ/Ϫ mice versus wild type p53 controls. p53 Ϫ/Ϫ mice demonstrated increased aortic plaque formation, with increased rates of cell proliferation and reduced rates of apoptosis in brachiocephalic arteries. Although most proliferating cells were monocyte/macrophages, apoptotic cells were both vascular smooth muscle cells (VSMCs) and macrophages. Transplant of p53 bone marrow to p53 Ϫ/Ϫ /ApoE Ϫ/Ϫ mice reduced aortic plaque formation and cell proliferation in brachiocephalic plaques, but also markedly reduced apoptosis. To examine p53 regulation of these processes, we studied proliferation and apoptosis in macrophages, bone marrow stromal cells and VSMCs cultured from these mice. Although endogenous p53 promoted apoptosis in macrophages, it protected VSMCs and stromal cells from death, a hitherto unknown function in these cells, in part by inhibiting DNA damage response enzymes. p53 also inhibited stromal cell expression of VSMC markers. We conclude that endogenous levels of p53 protect VSMCs and stromal cells against apoptosis, while promoting apoptosis in macrophages, and protect against atherosclerosis development. (Circ Res. 2005;96:667-674.)

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