Abstract 3384: p66shc Deletion Rescues EPC Deficit Induced by High Glucose
Reduced Endothelial Progenitor Cell (EPC) number and function are relevant components of the angiogenesis impairment observed in diabetic patients. A close relationship exists between hyperglycemia, oxidative stress and diabetic complications. In fact, high glucose determines the overproduction of reactive oxygen species (ROS) by the mitochondria. p66Shc gene regulates the apoptotic responses to oxidative stress. Indeed, p66Shc−/− mice display decreased ROS production and increased resistance to ROS-induced cell death in a variety of patho-physiologic settings. We examined whether EPC deficit induced by high glucose was rescued in EPC derived from p66Shc−/− mice. EPC number was assessed by measuring fibronectin-adhering cells that displayed Ac-LDL uptake and lectin binding and expressed CD34, VWF when cultured either in 50 mM glucose (HG) or in iso-osmotic control medium, for 7 days. EPC derived either from peripheral blood or from the c-kit+ fraction of the bone marrow displayed similar phenotypes. We found that HG induced a >5 fold increase in p66Shc protein expression and that HG exposure decreased wt EPC number by >40%, while p66Shc−/−EPC were not sensitive to HG inhibition. p66Shc−/− EPC resistance to HG was associated to reduced oxidative stress, as assessed using the red/ox sensitive probes DCHF and DHE. Indeed, HG significantly increased both DCHF+ and DHE+ cells in p66Shc wt population (>4 fold and >1.7 fold, respectively), while the −/− counterpart was unaffected. To functionally link ROS production to EPC response to HG, p66Shc−/− EPC were reconstituted either with p66Shc wt or with a p66Shc allele (p66Shc-qq) that was devoid of its ROS-generating function. We found that only wt p66Shc and not the qq mutant, rescued the −/− EPC sensitivity to HG. One major feature of oxidative stress is its the ability to reduce the bio-availability of nitric oxide (NO) that, in turn, plays a crucial role in EPC function. We found that the NO donor DETA-NO, rescued EPC deficit induced by HG in wt cells, while p66Shc−/− EPC became HG-sensitive upon treatment with L-NAME, a NO-synthase inhibitor. We conclude that p66Shc deletion rescues oxidative stress, NO and EPC deficit induced by HG, indicating a potential therapeutic target in diabetic vasculopathy.