ABSTRACT
Rac1, a subunit of NADPH oxidase, plays an important role in directed endothelial cell motility. We reported previously that Rac1 activation was necessary for choroidal endothelial cell migration across the retinal pigment epithelium, a critical step in the development of vision-threatening neovascular age-related macular degeneration. Here we explored the roles of Rac1 and NADPH oxidase activation in response to vascular endothelial growth factor treatment in vitro and in a model of laser-induced choroidal neovascularization. We found that vascular endothelial growth factor induced the activation of Rac1 and of NADPH oxidase in cultured human choroidal endothelial cells. Further, vascular endothelial growth factor led to heightened generation of reactive oxygen species from cultured human choroidal endothelial cells, which was prevented by the NADPH oxidase inhibitors, apocynin and diphenyleneiodonium, or the antioxidant, N-acetyl-L-cysteine. In a model of laser-induced injury, inhibition of NADPH oxidase with apocynin significantly reduced reactive oxygen species levels as measured by dihydroethidium fluorescence and the volume of laser-induced choroidal neovascularization. Mice lacking functional p47phox, a subunit of NADPH oxidase, had reduced dihydroethidium fluorescence and choroidal neovascularization compared with wild-type controls. Taken together, these results indicate that vascular endothelial growth factor activates Rac1 upstream from NADPH oxidase in human choroidal endothelial cells and increases generation of reactive oxygen species, contributing to choroidal neovascularization. These steps may contributed to the pathology of neovascular age-related macular degeneration.
Subject(s)
Choroid/blood supply , Choroidal Neovascularization/metabolism , Endothelium, Vascular/metabolism , NADPH Oxidases/metabolism , Pigment Epithelium of Eye/metabolism , Vascular Endothelial Growth Factor A/pharmacology , Acetylcysteine/pharmacology , Adult , Animals , Blotting, Western , Cell Movement/drug effects , Cells, Cultured , Choroid/metabolism , Choroidal Neovascularization/drug therapy , Choroidal Neovascularization/etiology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Free Radical Scavengers/pharmacology , Humans , Immunoenzyme Techniques , Lasers/adverse effects , Macular Degeneration/etiology , Macular Degeneration/metabolism , Macular Degeneration/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidases/antagonists & inhibitors , Onium Compounds/pharmacology , Phosphoproteins/physiology , RNA, Messenger/genetics , RNA, Small Interfering/pharmacology , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , rac1 GTP-Binding Protein/antagonists & inhibitors , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
PURPOSE: To investigate whether oxygen stresses experienced in retinopathy of prematurity (ROP) trigger signaling through reactive oxygen species (ROS) and whether the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway lead to intravitreous neovascularization (IVNV) in an oxygen-induced retinopathy (OIR) rat model. METHODS: Newborn rat pups exposed to repeated fluctuations in oxygen and rescued in supplemental oxygen (28% O(2), 50/10 OIR+SO) were treated with apocynin, an NADPH oxidase and ROS inhibitor (10 mg/kg/d), AG490, a JAK2 inhibitor (5 mg/kg/d), or phosphate-buffered saline. Intraperitoneal injections were given from postnatal day (P)12 to P17 (apocynin), or from P3 to P17 (AG490). Outcomes were intravitreous neovascularization and avascular/total retinal areas, vascular endothelial growth factor, phosphorylated JAK2, and phosphorylated STAT3. RESULTS: Apocynin significantly reduced phosphorylated STAT3 in 50/10 OIR+SO (P = 0.04), in association with previously reported inhibition of the IVNV area. Inhibition of JAK with AG490 significantly reduced phosphorylated JAK2 (P < 0.001), phosphorylated STAT3 (P = 0.002), and IVNV area (P = 0.033) in the 50/10 OIR+SO model compared with control. CONCLUSIONS: Activation of NADPH oxidase from supplemental oxygen works through activated STAT3 to lead to IVNV. In addition, inhibition of the JAK/STAT pathway reduces IVNV. Further studies are needed to determine the effects and relationships of oxygen stresses on JAK/STAT and NAPDH oxidase signaling.
