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X. Zhong, H. Huang, J.-K. Shen, L. Zentilin, M. Giacca, S. Vinores; The Effects of Overexpression of rAav9-vegf-b167 on Ocular Angiogenesis, Inflammation, and Blood-Retinal Barrier. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4492.
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© ARVO (1962-2015); The Authors (2016-present)
VEGF-B is probably the least studied VEGF family member. Its biological function remains debatable. This study explores the effects of overexpression of VEGF-B167 on ocular angiogenesis, inflammation, and the blood-retinal barrier (BRB).
Two recombinant adeno-associated virus serotype 9 (rAAV9) vectors were prepared, one expressingthe 167 amino acid isoform of mouse VEGF-B (rAAV9-VEGF-B167) and the other expressing the GFP reporter gene (rAAV9-GFP). Approximately 109 pfu rAAV9-VEGF-B167 or rAAV9-GFP was subretinally injected into 4-week-old C57BL/6J mice. The efficacy of AAV mediated gene transfer was assessed by real time quantitative PCR (qPCR) or by directly observing expression of the GFP control gene under fluorescence microscopy. Histological examinations were done to observe inflammation. The BRB was assessed using a quantitative assay with 3H-mannitol as a tracer. Oxygen-induced ischemic retinopathy (OIR) was induced by placing P7 mice, which received rAAV9-VEGF-B167, intravitreously in left eyes and rAAV9-GFP in right eyes at P5, in 75% oxygen for 5 days and removing them to room air for 5 days. The resultant retinal neovascularization (NV) was stained with GSA-lectin. Retinal NV was quantified with Photoshop software.
Under fluorescence microscopy, GFP expression was clearly demonstrated, primarily in the RPE and the outer retina, 1-6 weeks after delivery. qPCR indicated that there was a 5.7 fold higher mRNA expression of VEGF-B167 in the rAAV9-VEGF-B167group than in the rAAV9-GFP group. Endogenous VEGF-B was localized, primarily to retinal vessels. BRB assay showed that VEGF-B167 did not increase retinal vasopermeability compared to the control group (PRRLR=0.05,PRLLR=0.1). There was no evidence of an inflammatory response or vessel abnormality following injection of the rAAV9 vectors in normal adult mice. However, VEGF-B167 significantly increased pathological retinal NV in OIR.
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