Enhancement of the Anti-Apoptotic and Angiogenic Properties of Bone Marrow-Derived Endothelial Progenitor Cells by Bcl-2 Gene Transfer in Rats

Objective: To investigate the effects of Bcl-2 gene overexpression on apoptosis induced by hyperglycemic and hypoxic conditions in rat bone marrow-derived endothelial progenitor cells (EPCs). Methods: Primary EPCs were harvested from Sprague-Dawley rats, cultured in hyperglycemic and hypoxic conditions and modified by Bcl-2 gene via a lentivirus vector. Apoptosis of EPCs was evaluated by flow cytometry using Annexin V-FITC staining. The mRNA and protein levels of Bcl-2, NF-kappa B p65, Tie-2, JNK, Notch-1, p38MAPK, and VEGF-R2 in the EPCs were detected by RT-PCR and Western blotting. The in vivo effects of BCL-2 gene overexpression were analyzed in diabetic rats treated with allogenetic EPCs. Plasma VEGF levels and capillary densities in the treated animals were determined by ELISA and immunohistochemistry, respectively. Results: Culture in hyperglycemic and hypoxic medium increased apoptosis and induced expression of NF-kappa B p65, Tie-2, JNK, Notch-1, and p38MAPK genes, but inhibited Bcl-2 and VEGF-R2 gene expression in the EPCs at 24 h. Bcl-2 gene transfection reversed these effects in vitro. Administration of Bcl-2 gene-transfected EPCs in vivo increased plasma VEGF levels and capillary densities of the ischemic hind limbs at 14 days post treatment in the animals. Conclusions: Gene transfer of Bcl-2 can inhibit hyperglycemic and hypoxic-induced apoptosis of EPCs and promote angiogenesis by affecting angiogenic and oxidative pathways.