Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear1–6. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.

Abstract-Transplantation of bone marrow cells as well as circulating endothelial progenitor cells (EPC) enhancesneovascularization after ischemia. The chemokine receptor CXCR4 is essential for migration and homing of hematopoietic stem cells. Therefore, we investigated the role of CXCR4 and its downstream signaling cascade for the angiogenic capacity of cultured human EPC. Ex vivo, differentiated EPC derived from peripheral blood abundantly expressed CXCR4. Incubation of EPC from healthy volunteers with neutralizing antibodies against CXCR4 profoundly inhibited vascular endothelial growth factor-and stromal-derived factor-1-induced migration as well as EPC-induced angiogenesis in an ex vivo assay. Preincubation of transplanted EPC with CXCR4 antibody reduced EPC incorporation and impaired blood-flow recovery in ischemic hindlimbs of nude mice (57Ϯ4% of normal perfusion versus untreated EPC: 80Ϯ11%, PϽ0.001). Bone marrow mononuclear cells (BM-MNC) or EPC of heterozygous CXCR4 ϩ/Ϫ mice displayed reduced CXCR4 expression and disclosed impaired in vivo capacity to enhance recovery of ischemic blood flow in nude mice (blood flow 27Ϯ11% versus 66Ϯ25% using wild-type cells, PϽ0.01). Importantly, impaired blood flow in ischemic CXCR4 ϩ/Ϫ mice was rescued by injection of wild-type BM-MNC. Next, we investigated the role of CXCR4 for functional capacities of EPC from patients with coronary artery disease (CAD). Surface expression of CXCR4 was similar in EPC from patients with CAD compared with healthy controls. However, basal Janus kinase (JAK)-2 phosphorylation was significantly reduced and less responsive to stromal-derived factor-1 in EPC from patients with CAD compared with healthy volunteers, indicating that CXCR4-mediated JAK-2 signaling is dysregulated in EPC from patients with CAD. The CXCR4 receptor signaling profoundly modulates the angiogenic activity and homing capacity of cultured human EPC. Disturbance of CXCR4 signaling, as demonstrated by reduced JAK-2 phosphorylation, may contribute to functional impairment of EPC from patients with CAD. Stimulating CXCR4 signaling might improve functional properties of EPC and may rescue impaired neovascularization capacity of EPC derived from patients with CAD. (Circ Res. 2005;97:1142-1151.) Key Words: coronary artery disease Ⅲ endothelium Ⅲ angiogenesis Ⅲ EPC C irculating endothelial progenitor cells (EPC) play a crucial role in postnatal neovascularization. 1-3 Increasing evidence suggests that transplantation of cultureexpanded progenitor cells or bone marrow-derived progenitor cells successfully promotes therapeutic neovascularization in both ischemic hindlimbs as well as acute myocardial infarction models. 4 -7 Moreover, recent clinical pilot studies suggest that not only restoration of blood flow in peripheral artery disease but also functional regeneration and left ventricular remodeling can be enhanced after autologous transplantation of bone marrow-derived cells or cultured EPC in patients with coronary atherosclerosis. 8 -11 Further evidence indicates that no...

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...