Effect of PEDF on the Proliferation of Retinal Pigment Epithelial Cells and Pericytes

Abstract

Abstract: : Purpose:Diabetes mellitus and age-related macular degeneration (ARMD) account for major causes of blindness in industrialized countries. In diabetes, the proliferation of retinal endothelial cells is induced in part by progressive loss of pericytes, which is associated with lack of proliferation inhibitors such as TGFß2. In ARMD, the proliferation of choroidal endothelial cells is evoked by growth factor overproduction originating from proliferating retinal pigment epithelial cells (RPE). In this study we investigated the effect of PEDF, a potent serin protease inhibitor on bovine RPE and pericytes. Methods:BRPE und pericytes were isolated and cultured in DMEM+10% FCS. The cultures of RPE and pericytes were confirmed by immunohistochemistry for cytokeratin and α-smooth muscle actin respectively. Confluent 96-well plates were starved in serum-free medium. After 24 h, the cells were incubated with PEDF (0.1-100 ng/ml) in free serum medium containing [3H]-thymidine for another 24 h. A migration assay was performed by seeding BRPE to confluency in 6-well-multiplates and scraping the cells off from half the well with a cotton tip. The remaining cells were stimulated with PEDF (0.1-100 ng/ml) for 24 h. Subsequently, the cultures were fixed, stained and the migrating cells were counted. Statistical analysis was performed using two-sample t-test. Results:PEDF showed different effects on BRPE and on pericyte proliferation. PEDF directly inhibited the proliferation of BRPE at a concentration of 1 ng/ml (p<0.0002). Further, PEDF was able to inhibit BRPE migration at a concentration range between 0.1-100 ng/ml with maximal effect of 1 ng/ml (p<0.001). The proliferation of pericytes significantly increased in the presence of PEDF at the concentration of 10 ng/ml (p<0.0001). Conclusions:Our data shows that PEDF has a concentration-dependent effect and was able to inhibit the proliferation and migration of RPE and stimulate the proliferation of pericytes. This suggests a possible therapeutic scenario for proliferative eye disorders. First, PEDF could restore the inhibitory influence on endothelial cells through promoting pericytes proliferation. Second, PEDF inhibits the proliferation of RPE which would be associated with decreased amount of growth factors and results with decrease of endothelial cells proliferation.

Keywords: growth factors/growth factor receptors • retinal pigment epithelium • proliferation