Abstract 965: Prevention of Apoptosis in Endothelial Progenitor Cells Treated with the PPAR-γ Agonist Pioglitazone
Background: PPAR-γ agonists have been suggested to improve endothelial function in addition to insulin sensitizing. However, the underlying molecular mechanisms are incompletely understood. Bone marrow-derived endothelial progenitor cells (EPC) contribute to endothelial repair and neoangiogenesis.
Methods and Results: Treatment of C57/Bl6 mice with pioglitazone, 20 mg/kg/day, 10 days, increased circulating Sca-1/VEGFR-2 positive EPC to 235±60% of vehicle-treated mice (n=24 per group). EPC in the bone marrow were upregulated to 166±30 %. DiLDL/lectin positive EPC expanded from spleen-derived mononu-clear cells were upregulated to 231±21%. Upregulation of EPC was persistent after 20 days of treatment. Treatment with TZD increased the SDF-1-induced migratory capacity per number of EPC by 246±73% and enhanced in- vivo neoangiogenesis by 2-fold (disk assay, 214±42%, 20 d). Co-treatment with the NOS inhibitor L-NAME, 50 mg/kg, did not alter the upregulation of EPC by TZD. EPC from TZD-treated animals were characterized by reduced apoptosis (65±2.8 % of vehicle). To test these effects in humans, EPC were isolated from peripheral blood of healthy donors and cultured for 48h. Incubation of EPCs with H2O2 for 6 to 24 h resulted in a dose- and time-dependent increase of apoptosis. Pre-treatement with pioglitazone (10 μM, 24h) prevented H2O2-induced apoptosis (20.6±7.7% vs 46.4±11.3% apoptotic cells, Annexin-V FACS). Similarly, quantification of EPC apoptosis by DAPI staining showed TZD-mediated protection (19.3±10.4% vs 68.7±17.2% apoptotic cells). The inhibition of EPC apoptosis by TZD was abolished in the presence of the PI3K inhibitors LY294002 or wortmannin but not by LNMA. Expression of telomere repeat-binding factor 2 is a marker for replicative senescence. Treatment with TZD upregulated TRF2 expression in mouse EPC to 320±50 %. Similarly, TRF2 was increased in human EPC treated with pioglitazone, 1 μM, 72 h, to 344±36%.
Conclusion: The PPAR-γ agonist pioglitazone upregulates the number and functional capacity of EPC. Pioglitazone prevents apoptosis of EPC in mice as well as in human EPC in a PI3K-dependent but NO-independent manner. Reduction of EPC apoptosis by TZD may be a potential beneficial mechanism for patients with vascular diseases.