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, p22(phox), p47(phox), p67(phox), 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 p67(phox) 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.
Li
Su-min
SM
Department of Reproductive Medicine, University of California San Diego, La Jolla, California 92093, USA.
Zeng
Ling-wen
LW
Feng
Lin
L
Chen
Dong-bao
DB
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D008856:Microscopy, Fluorescence; D019255:NADPH Oxidases / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010920:Placenta / Q000098:blood supply / Q000502:physiology*; D011247:Pregnancy; D034741:RNA, Small Interfering; D012756:Sheep; D015398:Signal Transduction / Q000502:physiology; D013481:Superoxides / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D013481:Superoxides; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein