Aging and longevity genes p66shc and junD mediate age-related dysfunction of angiogenic early outgrowth cells: mechanistic insights into vascular repair

Abstract

Purpose: Cardiovascular disease is markedly age-dependent. Early outgrowth cells (EOCs) are important modulators of the vascular repair process, favouring myocardial neovascularization. Impairment of EOCs functionality in human aging is mostly driven by reactive oxygen species (ROS), but the molecular mechanisms remain largely unknown. We previously reported that transcription factor JunD and mitochondrial adaptor p66Shc are critically involved ROS-induced vascular aging. The present study investigates the role of JunD and p66Shc in age-related EOCs dysfunction. Methods: EOCs were isolated and cultured from peripheral blood mononuclear cells of young (24±4 years; n=5) and old (63±5 years; n=6) healthy volunteers enrolled via the blood donation service of the University Hospital Zurich, Switzerland. Gene silencing of p66Shc was performed with siRNA technology (Microsynth®), while JunD overexpression was obtained with a predesigned vector (Origene®). Scrambled siRNA or empty vector were used as negative controls for p66Shc and JunD, respectively. Three days after transfection young and old EOCs were harvested for measurement of O2levels by ESR spectroscopy, migration assay and real-time PCR. Written informed consent was obtained from all subjects. Results: EOCs isolated form old individuals showed higher p66Shc expression and JunD downregulation as compared with young subjects. p66Shc and JunD deregulation in old EOCs was associated with increased O2generation, blunted migration, upregulation of the NADPH subunit Nox2 as well as reduced expression of the scavenger enzymes manganese superoxide dismutase (MnSOD) and aldehyde dehydrogenase-2 (ALDH2). Interestingly, either p66Shc knockdown or JunD overexpression significantly suppressed age-related O2production, improved EOCs migration and restored the balance between oxidant and antioxidant enzymes. Conclusions: p66Shc and JunD are critically involved in age-dependent EOCs dysfunction by altering their redox state. Modulation of such aging and longevity genes restores normal repair capacities in EOCs from aged individuals and may be useful as an ex vivo strategy to improve the clinical efficacy of stem cell therapy in elderly cardiovascular patients.