Extracellular matrix production by cat retinal pigment epithelium in vitro: Characterization of type IV collagen synthesis

Abstract

Feline retinal pigment epithelial cells (RPE) produced an extracellular matrix (ECM) in vitro which was located between the basal surface of the RPE and the culture plate. This ECM had three morphological components: bundle, granular and fibrillar. After 14 days in culture the basal extracellular space contained small amounts of bundle material; granular and fibrillar material were infrequently observed at this time. The amount of ECM material increased with increasing time in culture. The accumulation of the granular component extracellularly was greatest between 60 and 108 days. Fibrillar material, although occasionally observed in the ECM, appeared to be an infrequent component. By 145 days, the ECM filled the extracellular space between the RPE and the culture plate. The time-dependent increase of the ECM indicated continued synthesis and secretion of ECM into the basal extracellular space by the RPE. Confluent RPE cultures, or choroidal/scleral fibroblasts, were incubated for 24 hr with [ 14C]-proline. Newly synthesized collagen, either in the culture medium or the cell layer, was co-precipitated with added carrier collagen by (NH 4) 2SO 4. The samples, with or without reduction and alkylation, were digested with pepsin and fractioned by selective salt precipitation and carboxymethyl(CM)-cellulose chromatography. The resulting fractions were further analyzed, or purified for thin layer chromatography (TLC) amino acid analysis, by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Cultured RPE cells, but not choroidal/scleral fibroblasts, produced labelled peptides which were characterized as α 1 (IV), and α 2 (IV) collagen chains by CM-cellulose chromatography, SDS-PAGE, proline:hydroxyproline ratios and sensitivity to bacterial collagenase. In contrast, choroidal/scleral fibroblasts produced labelled α 1 (I), β 12 (I) and α 2 (I) collagen chains. The synthesis of type IV collagen by RPE cells may reflect the production of ECM observed by electron microscopy in cultured feline RPE cells.