Abstract
Purpose: :
Connective tissue growth factor (CTGF) has been implicated as a key player in the pathogenesis of fibrosis, and CTGF immunoreactivity has been demonstrated in proliferative vitreoretinopathy membranes. We examined whether protein synthesis of CTGF occurs in hRPE cells. We also investigated whether pigment epithelium–derived factor (PEDF) inhibits protein synthesis of CTGF, since it has been shown to inhibit the actions of other growth factors.
Methods: :
Primary hRPE cultures were established from human eyes. Experiments were performed using test media which consisted of 1% Fetal Bovine Serum (FBS), 10% FBS, or 10% FBS + 0.01 µg/mL PEDF. F12 was used as a control. Cell proliferation was measured using 3–H thymidine incorporation, and cells were directly counted using the trypan exclusion method. 14C–CTGF protein was immunoprecipitated using the COOH–terminal of CTGF–specific antibody. Immunohistochemistry was also performed using the COOH–terminal of CTGF–specific antibody. Data were analyzed by the student "t" test, and are reported as mean±SEM. A p<0.05 was considered statistically significant.
Results: :
Increasing concentrations of FBS stimulated hRPE cell proliferation in a dose–dependent manner as determined by 3–H thymidine incorporation (3626±1530 vs. 5028±822 vs. 18,339±5383). PEDF inhibited FBS–stimulated hRPE cell proliferation, as monitored by 3–H thymidine incorporation (18,339.3±5383 vs. 12,182±3257.2, p<0.05). Direct cell counting also showed that PEDF inhibited FBS–stimulated hRPE cell proliferation (21,9375±3.9 vs. 13,9375±2.5, p<0.05). Increasing concentrations of FBS stimulated 14C–CTGF synthesis in a dose–dependent manner in hRPE cells (4743±1500 vs. 7261±2296 vs. 17,336.3±5779). PEDF inhibited FBS–stimulated 14–C CTGF synthesis in hRPE cells (17,336±5778 vs. 12,229.8±6408.2, p<0.05). Immunohistochemical studies showed decreased positive immunoreactivity of CTGF in hRPE cells exposed to PEDF.
Conclusions: :
CTGF protein synthesis occurs in hRPE cells and is stimulated by FBS. PEDF inhibits CTGF synthesis in FBS–stimulated cells, and also inhibits hRPE cell proliferation. Since proliferative vitreoretinopathy (PVR) is characterized by the formation of membranes, PEDF may be useful in treating proliferative eye disease.
Keywords: retinal pigment epithelium • proliferative vitreoretinopathy • signal transduction: pharmacology/physiology