Reactive Oxygen Species Might Have Important Roles On Angiogenesis of Endothelial Progenitor Cells Derived From HCB(Human Cord Blood) Which Are Stimulated by VEGF.

VEGF is a key angiogenic growth factor stimulating proliferation, migration, and tube formation on endothelial cells (ECs), that works through the VEGF receptor type 2 (VEGR2, KDR/Flk1). Reactive oxygen species (ROS) such as superoxide and H₂O₂ have roles signaling for molecules on angiogenesis. In present study, the aim is to investigate the roles of reactive oxygen species on neovascularization in endothelial progenitor cells.

Mononuclear cells isolated from UCB were cultured using EGM-2 medium with VEGF, IGF-1 and FGF or basal medium in presence or absence of VEGF for 7 days. Outgrowing endothelail progenitor cells (Yoo et al, STEM CELLS 21:228-235, 2003) at first week of culture were analyzed ROS production by dichlorofluorescein (DCF) fluorescence by use of 2,7-dichlorodihydro-fluoresceine-diacetate (H2DCF-DA). In order to determine that ROS production might involve in EPC proliferation and migration, we had analyzed the impact of N-acetyl-L-cysteine (NAC), broad spectrum ROS scavenger, and NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI) using the proliferation, in vitro tube formation matrigel assay, migration assay with SDF-1/VEGF. We also analyzed the expression of NOX2-based NADPH oxidase (gp91^phox) and activation of ERK2 and Akt (Thr308 and Ser473) using VEGF with or without DPI.

Intracellular ROS level were increased during endothelial progenitor cell culture and were higher in UCB compared to that of BM and increased by VEGF treatment. Proliferation, in vitro tube formation matrigel assay and migration assay on endothelial progenitor cells using SDF-1/VEGF were decreased with additions of ROS scavenger DPI when compared with that of control group. In western blot data, NOX2-based NADPH oxidase(gp91^phox) was increased by VEGF and decreased by addition of DPI. VEGF induced pERK2 expression was also decreased by DPI and that finding was correlated on down-regulation of endothelial cell proliferation by DPI. Activation of Ser473 Akt was found in control group and decreased by VEGF and rebounded by VEGF and DPI. But Thr308 Akt was not activated in our experiments.

These results suggested that NOX2-based NADPH oxidase(gp91^phox)-induced ROS might play important roles on EPCs migration and proliferation by VEGF. Namely, manipulating the level of ROS biochemically may alter the pathogenesis in cardiovascular and in ischemic limb diseases. In results, these data may be useful to develop new therapeutic strategies.

No relevant conflicts of interest to declare.