Insulin Upregulates VEGF and Aggravates Retinopathy of Prematurity in Newborn Rats

Abstract

Abstract: : Purpose: Upregulation of VEGF may be the key event in endothelial cell proliferation of diabetic retinopathy. Paradoxically initiation of insulin treatment sometimes coincides with aggravation of diabetic retinopathy. In the present study, we examined whether insulin upregulates VEGF in retinal and RPE cell culture. In addition, we studied the effect of insulin on an in vivo model of retinal neovascularization. Methods: Cultured rat retinal cells and human RPE cells were exposed to insulin (1µg/ml). Levels of VEGF, phospho–PKC and phospho–PI3 kinase, were measured by Western blots. To produce subclinical ROP, rat pups were raised in a 50 ± 5 % oxygen chamber from P7 to P12, and then raised in room air. Insulin (4 u/D) was given IP from P12–P17 together with glucose infusion. At P17, retinal vascular proliferation was assessed by the fluorescein dextran angiography. VEGF levels in retina were estimated by immunohistochemical staining. On transverse eyeball sections, extraretinal endothelial nuclei that were positive for factor VIII were counted. Results: Western blots showed that levels of VEGF in rat retinal and RPE cell cultures increased after 24 h treatment with 10–1000 ng/ml of human insulin. Addition of PI₃K inhibitor reduced the insulin–induced increase of VEGF. Under the condition that produces subclinical ROP, insulin administration induced preretinal endothelial cell proliferation and significant preretinal hemorrhage. Retinopathy scores increased from 5.5 to 7.5, and the number of nuclei extending beyond the ILM increased from 18 to 59. Immunohistochemistry revealed that VEGF level was increased in the inner nuclear and ganglion cell layers. Conclusions: Insulin treatment induced VEGF in retinal and RPE cell cultures as well as in a rat ROP model. Insulin also.increased endothelial cellular proliferation and aggravated retinal neovascularization in this in vivo model. This finding may explain the mechanism of worsening in diabetic retinopathy at the commencement of insulin therapy.

Keywords: diabetic retinopathy • ischemia • retinal neovascularization