Vascular Endothelial Growth Factor Response to Insulin-Like Growth Factor in Normoxic and Hypoxic Cell Culture

Abstract

Purpose:: Retinopathy of prematurity (ROP) is a potentially blinding disease of the eye caused by neovascularization of the retina. It has been well-demonstrated that vascular endothelial growth factor (VEGF) plays a role in stimulating this angiogenesis.¹ A recent report suggested that insulin-like growth factor (IGF) is significant in ROP as well.² Clinically, low IGF levels in preterm infants were correlated to an increased risk of ROP. It was suggested that IGF influences VEGF in a downstream fashion, affecting neovascularization but leaving VEGF levels unchanged.² However, an immortalized strain of retinal pigment epithelial (RPE) cells has clearly shown increased VEGF secretion in response to IGF.³ The purpose of this study is to evaluate the effect of IGF-1 on VEGF expression in the individual retinal cell types that express VEGF.

Methods:: Retinal Muller cells, RPE cells, retinal endothelial cells and brain astrocytes were each isolated as primary cultures from newborn rats. Cultures were incubated together concurrently with 25 - 625 ng/ml of recombinant IGF-1 in normoxia and hypoxia (95% N2, 5%CO2) for 24 hours. VEGF was then analyzed in the growth medium by ELISA. Cells were lysed and the protein level was used to standardize results for cell density within each plate.

Results:: Astroctyes exhibited a 5-fold increase in VEGF secretion in hypoxia (p<0.0001). IGF treatment caused a dose-dependent increase in astrocyte VEGF secretion in hypoxia (p=0.0007), but no change in normoxia. Primary retinal pigment epithelial cells showed a 2-fold increase in VEGF secretion in hypoxia as well as a dose-dependent increase with IGF treatment (p=0.0146). Muller cell cultures displayed a 15-fold VEGF induction by hypoxia (p<0.0001), but no statistically significant increase in secretion associated with IGF treatment. Endothelial cells produced very little VEGF, though there was a significant increase with hypoxia (p=0.027).

Conclusions:: IGF-1 induces VEGF production under hypoxic conditions in primary retinal pigment epithelial cells and astrocytes. Muller cells have the largest hypoxic response and produce the highest quantities of VEGF under hypoxia. Endothelial cells produce an insignificant amount of VEGF in relation to other retinal cell types in culture.References:1. Pierce EA, et al. Proc Natl Acad Sci USA. 1995;92:905-9.2. Hellstrom A, et al. Proc Natl Acad Sci USA . 2001; 98(10):5804-8.3. Punglia RS, et al. Diabetes. 1997;46:1619-26.

Keywords: retinopathy of prematurity • astrocyte • vascular cells