The MAP Kinase Pathway and VEGF are Involved in IGF-1-Stimulated Proliferation of Human Retinal Pigment Epithelial Cells (hRPE)

Abstract

 

Purpose:

 

Abnormal proliferation and metaplasia of human retinal pigment epithelial cells (hRPE) have been implicated in the development of proliferative vitreoretinopathy. The purpose of this study is to investigate the stimulation by insulin-like growth factor-1 (IGF-1) on the proliferation of hRPE and elucidate the role of MAP kinase in the IGF-1 signaling cascade.

 

Methods:

 

Human RPE cells were harvested from postmortem non-pathological eyes. Cellular proliferation in the presence of increasing concentrations of IGF-1 and IGF-1 + PD98059 (an inhibitor of MAP kinase) was measured by ³H-thymidine incorporation and trypan blue exclusion (T) studies. Under the same experimental conditions, synthesis of vascular endothelial growth factor (VEGF) was measured utilizing ¹⁴C-methionine immunoprecipitation and immunocytochemical methods. Data were analyzed using Microsoft Excel.

 

Results:

 

IGF-1 stimulates hRPE cellular proliferation in a dose-dependent manner as demonstrated using ³H-thymidine incorporation and T. The maximal effect of IGF-1 was observed at a concentration of 50 ng/ml. There was also a concentration-dependent IGF-1-induced increase in VEGF synthesis demonstrated by ¹⁴C-methionine-VEGF immunoprecipitation. This was qualitatively confirmed by immunocytochemical staining using anti-VEGF and rhodamine-conjugated anti-rabbit IgG. Again, the maximal effect of IGF-1 was seen at 50 ng/mL. PD98059 suppressed both IGF-1-induced cell proliferation (694.58 ± 81.48 vs. 6208.50 ± 1252.62, CPM ± SEM, n = 8, p < 0.001) as well as IGF-1-stimulated VEGF production (967.98 ± 151.22 vs. 2997.58 ± 588.87, CPM ± SEM, n = 8, p < 0.05). Cells remained viable and healthy after exposure to PD98059 as determined by T.

 

Conclusions:

 

These studies suggest that IGF-1 is a mitogen for hRPE cells, possibly by stimulating synthesis of the angiogenic factor VEGF. Additionally, PD98059 inhibits IGF-1-induced production of VEGF and hRPE proliferation, suggesting that the MAP kinase pathway is involved in IGF-1-mediated angiogenesis.  

 

Keywords: retinal pigment epithelium • growth factors/growth factor receptors • proliferative vitreoretinopathy