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O. Konopatskaya, S.J. Harper, D.O. Bates, A. Churchill; The Expression of a Family of Inhibitory Splice Variants of VEGF in Human Ocular Tissues and Retinal Pigmented Epithelial Cells: Hypoxia Effect . Invest. Ophthalmol. Vis. Sci. 2005;46(13):456.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Vascular Endothelial Growth Factor (VEGF) controls pathological angiogenesis and the increase in vascular permeability in diabetic retinopathy and age–related macular degeneration, and is up–regulated by glucose in retinal cells (Sone et al., BBRC.1996; 221:193–8). The inhibitory splice variant VEGF165b is down–regulated in angiogenic tumours (Bates et al., Cancer Research 2002;62:4123–31) and is anti–angiogenic in vivo. We set out to determine whether VEGF165b is expressed in human ocular tissues and retinal pigment epithelial (RPE) cells and to investigate the effect of hypoxia on VEGF165b in RPE. Methods: Human eyes were obtained within 10–30 hours post–mortem from donors and ocular tissues dissected. RPE cells were isolated and characterised by immunohistochemistry with an anti–cytokeratin antibody. Western blotting and isoform–specific ELISA were utilized to examine VEGF protein expression, and RT–PCR to study VEGF mRNA. Results: Expression of VEGF165b was revealed in the lysate of RPE cells, retina, iris, lens, sclera and vitreous (n=2–4) by Western blotting with a C–terminal specific antibody. Multiple bands were detected. These were confirmed to be VEGF isoforms by Western blotting with a commercial pan–VEGF antibody. This suggests that multiple VEGFxxxb isoforms were present, eg VEGF121b VEGF189b, VEGF148b and large VEGF. Furthermore, RT–PCR of RPE RNA using primers flanking the 3’UTR and exon 7 of the mRNA, showed VEGFxxxb mRNA expression. Hypoxia had no effect on steady–state levels of total VEGF and VEGFxxxb protein expression in the lysate of cultured human RPE, as Western blotting showed. However, hypoxia induced upregulation of secreted levels of total VEGF in conditioned medium, determined by ELISA (3.7±0.86 fold increase over normoxic control after 48 hrs, n=3, P<0.005), and did not increase VEGFxxxb concentration. Conclusions: Human ocular tissues and RPE express multiple VEGFxxxb splice variants. The balance of stimulatory and inhibitory isoforms may play a significant role in the development of angiogenic eye diseases. Funded by Diabetes UK.
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