PYRUVATE SUPPLEMENTATION ENHANCES VASCULAR ENDOTHELIAL GROWTH FACTOR PRODUCTION BY BONE MARROW-DERIVED MONONUCLEAR CELLS

1) Bone marrow‑derived mononuclear cells (BMMNC include endothelial progenitor cells (EPC which are character‑ ized by their secretion of angiogenic factors such as vascular endothelial growth factor (VEGF to recruit local endo‑ thelial cells thereby enabling the establishment of new blood vessels. Implantation of BMMNC has been clinically used for therapeutic purposes in the treatment of critical limb ischemia (CLI; results showed that it was ineffective in a substantial number of cases. To evaluate the appropriate concentration of pyruvate to achieve the highest VEGF gene expression cells were cultured with pyruvate at final concentrations up to 20 mM in 5% CO2 for 2‑4 days. The intracellular concentration of pyruvate was measured enzymatically and cell number and viability were determined. Expression levels of VEGF genes and numbers of CD31 + CD34 + cells were evaluated. Finally VEGF levels in the con‑ ditioned medium were examined in each condition. Pyruvate supplementation in murine BMMNC cultures success‑ fully increased intracellular pyruvate levels in a concentration‑dependent manner and 5 mM pyruvate was found to be the most appropriate to maintain viable cell number and up‑regulate VEGF gene after 2‑day culture. In addition VEGF in the conditioned medium was significantly elevated by the use of 5 mM pyruvate after 4‑day culture. From these results we suggest that preconditioning of BMMNC with 5 mM pyruvate for 2 days may be a useful way to safely and inexpensively enhance the angiogenic properties of BMMNC and the therapeutic effectiveness of cellular therapy for CLI.