TGF beta secretion modulates the density-dependent growth of pig retinal pigment epithelium in vitro.
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-beta neutralizing antibody (0.1-100 microg/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-beta (0.1-5 microg/ml). Blocking greater amounts of TGF-beta in the medium with higher doses of antibody (>10 microg/ml) also inhibited the growth of the newly plated RPE, in the presence or absence of a neighboring confluent cell layer.


CONCLUSIONS
The TGF-beta family of cytokines mediates the density-dependent growth suppression of RPE in vitro. Neutralizing the effect of these cytokines by adding anti-TGF-beta antibodies can result in more rapid growth of the RPE in vivo.
Tezel
T H
TH
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA.
Del Priore
L V
LV
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002417:Cattle; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D066298:In Vitro Techniques; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000254:growth & development*; D013552:Swine; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000276:immunology / Q000502:physiology*
D000906:Antibodies; D003470:Culture Media; D016212:Transforming Growth Factor beta