Abstract
Purpose: :
Acute intensive insulin therapy may worsen proliferative diabetic retinopathy. Since erythropoietin (Epo) plays an important role in neovascularization, we investigated the role of the RAS/Raf/ERK kinase signaling pathways in regulating Epo synthesis in cultured human retinal pigment epithelial (hRPE) cells.
Methods: :
hRPE cultures were established from three human eyes. hRPE cell proliferation was quantitated by 3H-thymidine incorporation (3H-thy). hRPE cells were treated with varying concentrations of insulin. Cells were labeled with 14C-methionine. Anti-Epo was used for immuno-precipitation of 14C-Epo and immuno-histochemical analysis. The insulin treated cells were also exposed to PD98059, a selective inhibitor of MAP kinase (also known as MAPK/ERK kinase), to mevastatin, an inhibitor of RAS signaling pathway, and to a RAF inhibitor (RafI). Data were analyzed by Student 't' test.
Results: :
Increasing concentrations of insulin stimulate hRPE proliferation as determined by 3H-thy. Insulin (0-5 µg/ml) stimulates 14C-Epo synthesis in a dose-dependent manner. Insulin (5 µg/ml) stimulated 14C-Epo synthesis was inhibited by mevastatin (30µM) (1215.38±370.87 vs.180.12±47.41, CPM±SEM, p<0.006, n=7), by RafI (1215.38±370.87 vs.611.64±220.18, CPM±SEM, p<0.04, n=7) and by PD98059 (25 µM) (3258.31±1547.15 vs.2622.30 ±1641.29, CPM±SEM, p<0.04, n=4). Immuno-histochemical studies showed an increase in Epo immuno-reactive cells in presence of insulin (5 µg/ml) when compared to controls and to cells exposed to insulin supplemented with PD98059 (25 µM).
Conclusions: :
Insulin stimulates Epo synthesis in hRPE cells and this synthesis appears to be mediated by the RAS/Raf/ERK kinase signaling pathway. Understanding of the complex regulation of Epo synthesis may provide useful information that will contribute to the development of therapeutic pharmaco-therapy to treat or prevent proliferative eye diseases.
Keywords: retinal pigment epithelium • proliferation • proliferative vitreoretinopathy