Effect Of Pedf In Combination With Growth Factors On Proliferation Of Bovine Retinal Endothelial Cells

Abstract

Abstract: : Purpose:Proliferative retinopathies such as diabetic retinopathy or age-related macular degeneration account for major causes of blindness. One of the possible targets in the therapy of these disorders is neovascularization, which is closely associated with IGF-1-, VEGF- and bFGF-induced endothelial cell proliferation. Recently, the novel serine protease inhibitor PEDF was shown to have strong antiproliferative effects on retinoblastoma cells. In this study we investigated if PEDF has an antiproliferative effect on IGF-1-, VEGF- and bFGF- induced proliferation of bovine retinal microvascular endothelial cells (BREC). Methods:BREC were isolated using the dynabeads-based method and cultured in ECGM-MV medium. Subsequently, BREC were detached with trypsin and seeded into 96-well-plates. Upon reaching confluency medium was changed to serum free defined medium containing (₃H)-thymidine and the cells were treated with PEDF alone at the concentration range of 0.1 to 100 ng/ml or with IGF-1 (50 ng/ml), bFGF (10 ng/ml) and VEGF (20 ng/ml) alone or in combination with PEDF (1 ng/ml) and PEDF alone (1 ng/ml) for 24 h. (₃H)-thymidine incorporation as a direct measure of BREC proliferation was assessed by liquid scintillation counting. In all experiments untreated serum free negative controls were used. Statistical analysis was performed using paired student's t-test. Results:PEDF showed different effects on BREC proliferation. PEDF directly inhibited the proliferation of BREC at the concentration of 1 ng/ml (p<0.001). The proliferation of BREC significantly increased (p<0.01) in the presence of PEDF at the concentration of 100 ng/ml. The concentrations of 0.1 and 10 ng/ml of PEDF had no effect. As compared to untreated control PEDF at the concentration of 1 ng/ml showed significant inhibition of bFGF, VEGF and IGF-1 induced proliferation (p<0.001). Conclusion:Our data show that PEDF alone has a concentration-dependent modulatory effect on the proliferation of bovine retinal microvascular endothelial cells. Additionally, PEDF is able to inhibit the proliferation of endothelial cells induced by VEGF, IGF-1 and bFGF in vitro. This promising data show that the anti-proliferative properties of PEDF can be of possible therapeutic value for pathologic ocular neovascularization.

Keywords: 566 retinal neovascularization • 423 growth factors/growth factor receptors