Abstract 775: p66Shc Deletion Rescues EPC Differentiation Deficit Induced By High Glucose
Background. Reduced endothelial progenitor cell (EPC) differentiation is one relevant component of angiogenesis impairment 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 differentiation deficit induced by high glucose was rescued in EPC derived from p66Shc−/ − mice.
Methods and results. EPC differentiation was assessed measuring the number of fibronectin-adhering cells that displayed Ac-LDL uptake and expressed Von Willebrand factor, but not Mac3 when cultured either in 50 mM glucose (HG) or mannitol (M), for 7 days. In peripheral-blood EPC, HG significantly decreased the efficiency of wt EPC differentiation (M=68.3%±1.8; HG =46.3%±3.3; p<0.001). Conversely, p66Shc−/ − EPC were resistant to HG differentiation inhibition (M=67.0%±3.2; HG =68.3%±2.7; p= ns). Similar results were obtained analyzing endothelial differentiation potential of c-kit+ cells derived from the bone marrow of wt and p66Shc−/ − mice. Interestingly, when bone marrow c-kit+ cells were grown in cytokine-rich medium, HG inhibited wt cell proliferation (fold increase at day 4, M=3.8±0.6, HG=2.5±0.6; p<0.0002), while p66Shc−/ − cKit+ cells were not affected (fold increase at day 4, HG=2.0±0.2, M=2.2±0.5; p=ns). The % of cKit+ cells throughout the expansion time course did not show significant differences between wt and , p66Shc−/ −. To assess whether EPC functional rescue was associated to reduced oxidative stress, differentiated EPC were incubated with the red/ox sensitive probe DCHF. It was found that HG increased DCHF+ cells in p66Shc wt population (fold increase=3.7±0.5; p<0.0001), while the −/ − counterpart was unaffected (fold increase=1.3±0.3; p=ns).
Conclusions. p66Shc deletion rescues oxidative stress and EPC differentiation deficit induced by HG, indicating a potential therapeutic target in diabetic vasculopathy.