TGF Beta Secretion Modulates the Density-Dependent Growth of Pig Retinal Pigment Epithelium in vitro

Abstract

Background: We aimed to identify the cytokine(s) responsible for the density-dependent growth regulation of pig retinal pigment epithelium (RPE) in vitro. Methods: Confluent monolayers of primary pig RPE were established on bovine corneal endothelial extracellular matrix-coated tissue culture well inserts wrapped with dialysis membranes with different molecular weight cutoffs (0.5–50 kDa). These confluent RPE monolayers were then cocultured with first passage porcine RPE plated at a density of 1 cell/mm², so that the newly plated RPE was bathed with different molecular weight fractions of the confluent cell media. Growth rates of the newly plated RPE were determined 72 h after plating and the molecular weight fraction of the confluent cell medium that inhibits the RPE proliferation was determined. First passage pig RPE (1 cell/mm²) were cocultured with confluent monolayers of primary pig RPE on inserts in the presence of different amounts of TGF-β neutralizing antibody (0.1–100 µg/ml). Growth rates of the newly plated RPE were calculated 72 h after plating to determine the antibody concentration that would maximize the growth rate of the newly plated RPE in the presence of an adjacent confluent RPE monolayer. Results: The growth rate of the newly plated RPE decreased when RPE were bathed with the 10- to 25-kDa fractions of medium from an adjacent confluent RPE monolayer. This growth inhibition reached statistical significance with the 25- to 50-kDa fractions (p < 0.05), and was abolished by adding pan-specific neutralizing antibody against TGF-β (0.1–5 µg/ml). Blocking greater amounts of TGF-β in the medium with higher doses of antibody (>10 µg/ml) also inhibited the growth of the newly plated RPE, in the presence or absence of a neighboring confluent cell layer. Conclusion: The TGF-β family of cytokines mediates the density-dependent growth suppression of RPE in vitro. Neutralizing the effect of these cytokines by adding anti-TGF-β antibodies can result in more rapid growth of the RPE in vivo.