Polarized Retinal Pigmented Epithelial Cell Cultures Treated With Connective Tissue Growth Factor Develop Extracellular Matrix-Containing Membranes

Abstract

Purpose: : In proliferative vitreoretinopathy (PVR), retinal pigmented epithelial cells (RPE) participate in the formation of contractile, smooth muscle actin (SMA) positive, cellular fibrovascular membranes with abundant extracellular matrix (ECM) on both surfaces of the retina. The purpose of this study was to establish conditions in which polarized RPE would develop a membrane rich in ECM components.

Methods: : Early passage RPE cells isolated from human fetal eyes were grown in a RPE differentiation media modified from that previously described by Hu and Bok (Mol Vis. 2001; 7:14-9) for approximately 1 month. Cells were trypsonized and 200 x10³ cells were seeded onto Transwell inserts (0.4um pore size) coated with fibronectin. Differentiated monolayer cultures maintained a transepithelial resistance (TER) greater than 200 ohms.cm², and were highly polarized. Polarized monolayers were treated with tumor necrosis factor-alpha (TNF-α; 10ng/ml) and/or connective tissue growth factor (CTGF; 30ng/ml) twice a week for 3 weeks. Control wells had media alone. Cultures were evaluated for TER (2x/week after treatment), and for expression of tight junction proteins collagen IV, smooth muscle actin and fibronectin by confocal immunofluorescent microscopy.

Results: : Polarized RPE monolayers treated with TNF-α + CTGF showed a decrease in TER from approximately 200 to 100 ohms.cm²; TER remained at this level over the 3 week treatment period. In the controls, TER increased steadily from approximately 200 to over 800 ohms.cm² over the 3 week period. RPE cells in the TNF-α + CTGF group showed a de-differentiated epithelial phenotype with decreased staining for tight junction associated protein ZO-1, and its functional correlate, a decrease in TER. Increased staining for SMA, and surface membrane formation with abundant immunoreactivity for collagen IV and fibronectin was present only with exposure to both agents. Control cultures or those treated with TNF-α or CTGF alone had little membrane formation and much less staining of ECM components.

Conclusions: : Polarized human RPE cultures treated with TNF-α + CTGF develop de-differentiated phenotypic changes and membranes on their surface demonstrating features of those seen in PVR. This in vitro model may be useful to study membrane formation in more detail.

Keywords: proliferative vitreoretinopathy • extracellular matrix • immunohistochemistry