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

Abstract

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.

NADPH oxidase 2 plays a role in experimental corneal neovascularization

“…NADPH oxidase has been shown to mediate processes that are important to pathological angiogen- esis including proliferation and migration of endothelial cells [48]. Inhibiting the activity or expression of Nox2 has indeed been demonstrated to attenuate the proliferation and migration of endothelial cell under the stimulation of Vegf [15,49]. Nox2 has also been found to be involved in the switching of endothelial cell phenotype from quiescent to pro-inflammatory in association with Vegf-A and angiopoietin-2 [50].…”

“…Several isoforms of the Nox subunit have been identified and the prototypical Nox is composed of membrane-bound Nox2 and p22phox subunits, as well as the cytosolic subunits p40phox, p47phox, p67phox and the small GTPase Rac (Supplementary Figure S1). There is evidence that VEGF can act to increase ROS generation by activating the catalytic domain of Nox2 [15]. Furthermore, it is known that Nox2 can also trigger VEGF signalling to regulate angiogenesis and vascular permeability via up-regulation of VEGF expression [16][17][18] as well as activation of its receptor, KDR/Flk-1 (VE-GFR2) [19].…”

Behind the Link between Copper and Angiogenesis: Established Mechanisms and an Overview on the Role of Vascular Copper Transport Systems

“…ATP7A, but not ATP7B, expression has been detected in foetal endothelia within the cytoplasm and at the baso-lateral membrane of the syncytiotrophoblast, where the ATPase regulates the absorption of copper from the placenta [138]. The role of ATP7A in placental angiogenesis is underpinned by the assumption that down-regulation of Rac-1 GTPase, directly recruited by the pump to the edges of protruding endothelial lamellipodia, negatively interferes with VEGF-activated pathways, as observed in ovine foeto-placental artery endothelial cells [139]. By virtue of its copper export activity, ATP7A potentially plays a crucial role in vascular morphogenesis.…”

“…In fact, ATP7A (but not CTR1 and ATOX1) knockdown destabilizes the endothelial junctions by the disruption of a scaffold assembly involving both the ATPase itself and the cytoskeletal protein IQGAP1 (IQ motif-containing GTPase activating protein 1) [151,152], interacting with phosphorylated (active) VEGF receptor 2 within the endothelial lamellipodia [153]. Interestingly, VEGF signalling in endothelial cells requires IQGAP1 to bind the small Rho GTPase Rac1 [153], an NADPH oxidase co-factor, contributing the growth-factor-driven angiogenesis by mechanisms not yet fully identified [139,154,155,156,157]. The structural nature of the contribution of ATP7A to the angiogenic processes has been corroborated by other authors' observations referred to VSMCs subjected to pro-angiogenic stimuli [118] (fig.…”

Antiangiogenic-Induced Hypertension: The Molecular Basis of Signaling Network

“…The Rac1/NOX-based system is present in placental endothelial cells [53]. VEGF elicits a burst of reactive oxygen species (ROS) through a NOX-mediated dimerization of CD146 (MCAM, melanoma cell adhesion molecule) [54].…”