Abstract
Purpose: :
Human retinal pigment epithelial cells (hRPE) have been implicated in the pathogenesis of several ocular diseases, including proliferative diabetic retinopathy (PDR). Erythropoietin (Epo), which has been previously identified in hRPE cells, has been shown to have an angiogenic potential equivalent to that of VEGF. Since very little is known about the effect of glucose on Epo production, we investigated the mechanism by which elevated glucose levels influence Epo synthesis in cultured hRPE cells.
Methods: :
Primary hRPE cell cultures were established from donor human eyes obtained from the Michigan Eye Bank. Cell viability and proliferation were assessed by the trypan blue exclusion method, and cell proliferation was also measured by 3H-thymidine incorporation. Epo-specific antibody was used to measure intracellular Epo synthesis, which was quantified by immunoprecipitation of 14C-methionine labeled Epo and also qualitatively visualized and localized by immunohistochemistry. Genistein, a tyrosine kinase inhibitor, was used to study the role of protein tyrosine kinases in Epo synthesis. Data were analyzed by Student’s t-test.
Results: :
Fetal Bovine Serum (FBS) stimulated hRPE cell proliferation in a dose-dependent manner as determined by 3H-thymidine incorportation, verifying a viable hRPE cell model that responds to biological stimuli. Glucose (0-20 mM) increased hRPE cell proliferation in a dose-dependent manner, as determined the trypan blue exclusion method. Glucose (0-10 mM) also stimulated 14C-methionine-Epo synthesis in hRPE cells in a dose-dependent manner. However, in the presence of genistein, the stimulatory effects of glucose on both cell number (30.5±4.66 vs. 24.75±2.72, n=4, p<0.05) and Epo synthesis (606±49 vs. 434±61, n=9, p<0.05) were inhibited. Immunohistochemical analysis confirmed that glucose stimulated Epo synthesis while the addition of genistein inhibited glucose-stimulated Epo production.
Conclusions: :
Glucose stimulates hRPE cell proliferation and increases Epo production. Glucose-stimulated Epo production may be mediated by a tyrosine kinase signaling pathway. These findings suggest that tyrosine kinase inhibitors may be of potential therapeutic value in the treatment of PDR.
Keywords: diabetic retinopathy • retinal pigment epithelium