Rac1-Dependent Intracellular Superoxide Formation Mediates Vascular Endothelial Growth Factor–Induced Placental Angiogenesis in Vitro

Vascular endothelial growth factor (VEGF) is one of the best characterized angiogenic factors controlling placental angiogenesis; however, how VEGF regulates placental angiogenesis has not yet completely understood. In this study, we found that all the components of assembling a functional NADPH oxidase (NOX2, p22phox, p47phox, p67phox, and Rac1) are expressed in ovine fetoplacental artery endothelial cells (oFPAECs) in vitro and ex vivo. Treatment with VEGF (10 ng/ml) rapidly and transiently activated Rac1 in oFPAECs in vitro and increased Rac1 association with p67phox in 5 min. Intracellular superoxide formation began to significantly increase after 25–30 min of VEGF stimulation, which was mediated by both VEGFR1 and VEGFR2. VEGF also stimulated oFPAE cell proliferation and migration and enhanced the formation of tube-like structures on Matrigel matrix. In oFAPEC transfected with specific Rac1 small interfering RNA (siRNA, 40 nm), VEGF-induced intracellular superoxide formation was completely abrogated in association with a 78% reduction of endogenous Rac1. In oFPAE cells transfected with the specific Rac1 siRNA, but not with transfection reagent alone or scrambled control siRNA, VEGF-induced cell proliferation, migration, and tube-like structure formation were dramatically inhibited. Pretreatment of an NADPH oxidase inhibitor apocynin also abrogates the VEGF-stimulated intracellular superoxide production and DNA synthesis in oFPAECs. Taken together, our results demonstrated that a Rac1/Nox2-based NADPH oxidase system is present in placental endothelial cells. This NADPH oxidase system appears to generate the second messenger superoxide that plays a critical role in the signaling control of the VEGF-induced placental angiogenesis.