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Xixiang Tang, Joshua Jianxin Wang, Sarah Xin Zhang; Role of Endothelial Nox4 in Retinal Vascular Development and Pathological Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4062.
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© ARVO (1962-2015); The Authors (2016-present)
NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidase in vascular endothelial cells. Our previous study has demonstrated that deletion of retinal Nox4 using small interference RNA reduces retinal neovascularization (NV) in oxygen-induced retinopathy (OIR); however, the role of endothelial Nox4 in retinal vascular development and pathological angiogenesis remains elusive. Here, we generated endothelial cell-specific Nox4 knockout (Nox4EC-KO) mice and investigated the role of endothelial Nox4 in retinal angiogenesis.
Nox4EC-KO mice were generated by crossing Nox4 floxed mice with Tie-2 Cre mice. Successful deletion of Nox4 in endothelial cells was confirmed by qPCR and western blot analysis using isolated brain microvascular endothelial cell (BMEC) from NoxEC-KO mice. Development of the retinal vasculature was evaluated by Isolectin B4 (IB4) staining of retinal whole mounts at postnatal days 7 (P7), P12 and P17. The effect of endothelial Nox4 deletion on vascular obliteration and pathological retinal NV was assessed in OIR at P9 and P17. Endothelial tip cells and signaling pathways of angiogenesis were examined by in vitro and in vivo studies.
Compared to wild type (WT) mice, neonatal Nox4EC-KO mice exhibited reduced radial expansion of superficial vascular plexus, lower retinal vascular area, and fewer vascular branching points in the retina at P7. The number of tip cell with filopodia bursts in the front sprouting vessels was significant decreased in Nox4EC-KO mice. Further, the tip cells appeared to be disoriented with sparser, shorter, and thinner filopodia in the KOs. Analysis of the deep and intermediate layers of retinal blood vessels showed a delayed development and reduced vascular density in Nox4EC-KO at p12 and p17, respectively. In OIR, the Nox4 EC-KO mice developed less severe retinal NV while had no effect on vaso-obliteration. Mechanistically, Nox4 deletion significantly reduced expression of Vegf, Vegfr2, Cxcr4, Cdh5, Ang1, Ang2, eNOS, and Itga5, and decreased protein levels of VEGF and p-VEGF-R2 in BMECs.
Taken together, our data indicate that endothelial Nox4 is implicated in retinal vascular development and contributes to hypoxia-induced pathological retinal angiogenesis. Targeting endothelial Nox4 may represent a promising therapeutic approach for retinal neovascular diseases such as proliferative diabetic retinopathy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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