p66ShcA modulates oxidative stress and survival of endothelial progenitor cells in response to high glucose

A close relationship exists between hyperglycaemia, oxidative stress, and diabetic complications. In fact, high glucose (HG) determines the overproduction of reactive oxygen species (ROS) by the mitochondria. p66(ShcA) is a gene that regulates the apoptotic responses to oxidative stress. Indeed, p66(ShcA) knockout (ko) mice display decreased ROS production and increased resistance to ROS-induced cell death in a variety of pathophysiological settings. Reduced endothelial progenitor cell (EPC) number, differentiation, and function are relevant components of the angiogenesis impairment observed in diabetic patients. We examined the role of p66(ShcA) in the EPC deficit induced by HG. Mouse bone marrow-derived c-kit(+) cells differentiate in endothelial-like cells when plated on fibronectin (BM-derived EPCs). We found that cell culture in the presence of HG up-regulated p66(ShcA) protein expression and that HG exposure markedly decreased the number of BM-derived EPCs. Conversely, p66(ShcA) ko BM-derived EPCs were not sensitive to HG inhibition. Indeed, the resistance of p66(ShcA) ko BM-derived EPCs to HG was associated with reduced levels of both apoptosis and oxidative stress. To functionally link the HG response to ROS production, p66(ShcA) ko BM-derived EPCs were reconstituted either with p66(ShcA) wild-type (wt) or with a p66(ShcA) allele (p66(ShcA) qq) that was devoid of its ROS-generating function. We found that only p66(ShcA) wt and not the qq mutant rescued p66(ShcA) ko cell sensitivity to HG. One major feature of oxidative stress is its ability to reduce the bio-availability of nitric oxide (NO) that, in turn, plays a crucial role in endothelial differentiation and function. We found that the p66(ShcA) deletion prevented the HG-induced increase of nitrotyrosine, and that the resistance to HG of p66(ShcA) ko BM-derived EPCs was prevented by NO synthase inhibition. With a reciprocal approach, the treatment of p66(ShcA) wt cells with a NO donor prevented the HG-induced deficit. Finally, using a Matrigel plug angiogenesis assay, we demonstrated that p66(ShcA) ko prevented diabetic impairment of angiogenesis in vivo. p66(ShcA) deletion rescues the BM-derived EPCs defect induced by HG, indicating p66(ShcA) as a potential therapeutic target in diabetic vasculopathy.