NADPH Oxidase Signaling Crucial for Neovascularization in Oxygen-induced Retinopathy in Mice: Nox2, Nox4 and VEGF Signaling

Abstract

Purpose: The proliferation of new blood vessels in the retina is a leading cause of vision impairment. NADPH oxidase (Nox) is involved in cell signaling for ischemia-induced angiogenesis, but its role in retinal neovascularization remains unclear. Recently we showed that the Nox2 isoform is involved in oxygen-induced retinopathy (OIR) in mice. We have now investigated the role of the Nox4 isoform, and show that a selective inhibitor of Nox4 expression reduces retinal neovascularization in this model.

Methods: In human umbilical vein endothelial cells (HUVEC) Nox isoforms and cell adhesion molecule expressions were evaluated by PCR. HUVEC proliferation and tubulogenesis were determined in a Matrigel medium. Neonatal wildtype and Nox4-/- mice aged 7d (P7) were exposed to 5d hyperoxia (75%O2), followed by 5d room air. Eyes were harvested on P8 and P17 for quantitation of retinal vaso-obliteration and neovascularization respectively. Retinal expression of Nox4 and VEGF-A were measured by PCR and superoxide anion generation was detected by in situ dihydroethidium staining of fresh frozen sections.

Results: Inhibition of Nox4 in vitro using either RNAi or a small molecule, specific inhibitor of Nox4 expression (2HF, 10-50μM) markedly reduced HUVEC proliferation and tubulogenesis. 2HF also abolished cytokine-induced ICAM-1 and VCAM-1 expression. In wildtype mice, OIR was characterized by central retinal vaso-obliteration at P8 and neovascularization at P17, which was associated with increases in Nox4 (12-fold), Nox2 (4-fold) as well as VEGF (2.5-fold) gene expression. At P17 the inner retina also showed superoxide generation and accumulation of macrophages and microglia. Nox4-/- mice exhibited markedly less retinal neovascularization (7.9+0.7%, n=13) but similar elevations of VEGF (2-fold) compared to wildtype (13.6+0.8%, n=14 and 2.5-fold respectively), in contrast to Nox2-/- mice where the VEGF increase was abolished. Analagous to Nox4-/-, in preliminary studies daily treatment with 2HF (1 mg/kg/d, ip) also reduced retinal neovascularization (by 30%) compared to vehicle-treated, wildtype control mice.

Conclusions: Clearly Nox4 facilitates retinal neovascularization in this model of OIR. Therapies targeting Nox4 could be of value to reduce aberrant retinal neovascularization in retinopathy of prematurity, diabetes and perhaps in the proliferative stages of macular degeneration.

Keywords: 700 retinal neovascularization • 706 retinopathy of prematurity • 634 oxidation/oxidative or free radical damage