Introduction: Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in patients with chronic obstructive pulmonary disease (COPD), an obstructive lung inflammatory disorder affecting approximately 20% of smokers. Even though numerous studies describe evidence of endothelial dysfunction in young healthy smokers and COPD patients, the molecular pathways that link cigarette smoke and CVD remain unclear. Increased DNA damage and cellular senescence have been recognized as important contributors to CVD. DNA damage, caused by factors such as oxidative stress, activates ataxia-telangiectasia mutated (ATM) kinase, a key player in the DNA damage response (DDR), and results in cell cycle arrest, senescence or apoptosis. Senescent vascular cells exhibit dysfunctional characteristics and have been shown to contribute to accelerating vascular aging and atherosclerosis. Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction also contributes to CVD. Blood outgrowth endothelial cells (BOEC, also called endothelial colony forming cells – ECFC), are a well characterized endothelial cell population with robust clonal proliferative potential and ability to form de novo vessels in vivo. This population has recently attracted considerable interest as a potential cell-based therapy for vascular regeneration, and as a tool to study endothelial dysfunction in patients. Our aim was to investigate whether EPC from smokers and COPD patients are dysfunctional due to increased DNA damage imposed by cigarette smoke-oxidative stress, which could contribute to the development of CVD, and to elucidate the pathways involved in this process. Methods: To investigate EPC dysfunction in smokers, we isolated and expanded BOEC from peripheral blood samples of healthy non-smokers, healthy smokers and COPD patients. The mononuclear fraction was placed in culture in the presence of endothelial growth factors and BOEC colonies appeared between days 7 and 24. BOEC colonies were expanded and used at passages 4 to 6 for all experiments. Endothelial senescence was measured by senescence-associated β-galactosidase (SA-β-Gal) activity. Expression of sirtuin (SIRT)-1, p16, p21, γ-H2AX and 53BP1 were measured by Western blotting and/or immunofluorescence confocal microscopy. SIRT1 activity was measured using a SIRT1 fluorescent activity assay kit. To investigate angiogenesis in vivo, BOEC were labelled with Vybrant DiI Cell-Labelling Solution, mixed with Matrigel and injected subcutaneously into the back of NOD.CB17-Prkdcscid/NcrCrl mice. Seven days later, the mice were sacrificed and the plugs were harvested and cryosectioned. Results: In vitro, BOEC from smokers and COPD patients showed increased DNA double-strand breaks (measured by γ-H2AX, 53BP1) and senescence (senescence associated-β-galactosidase activity, p16 and p21 levels) compared to non-smokers. Senescence negatively correlated with expression and activity of sirtuin-1 (SIRT1), a protein deacetylase that inhibits DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ATM kinase resulted in up-regulation of SIRT1 expression and decreased senescence. Interestingly, treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or a selective ATM inhibitor rescued the senescent phenotype. Using the in vivo Matrigel plug assay, BOEC from COPD patients displayed reduced ability to form capillary-like structures and increased DNA damage, senescence and apoptosis (measured by 53BP1, p16, TUNEL and cleaved-caspase 3 staining) compared to non-smokers. Conclusions: This study provides evidence of epigenetic alterations in endothelial progenitors from smokers, linked to cigarette smoke-oxidative stress, namely reduced SIRT1 expression via activation of the DNA damage response / ATM pathway. We demonstrated that progenitor cells of the endothelial lineage in smokers and COPD patients show reduced angiogenesis in vivo and display increased DNA damage and senescence, associated with reduced SIRT1 expression. These defects may contribute to endothelial dysfunction and cardiovascular events in smokers and COPD patients and could potentially constitute therapeutic targets for intervention.