Influence of Extracellular Membrane Proteins on RPE Functions

Abstract

Abstract: : Purpose: This study was designed to examine the role of basement membrane proteins on RPE function, specifically whether laminin, collagen Type IV and fibronectin modulate RPE cell attachment, phagocytosis and formation of junctional complexes as evidenced by transepithelial resistance (TER). Methods:: To measure cell attachment ARPE–19 cells were plated in 96–well plates coated with collagen type IV, human plasma fibronectin, or laminin at a density of 15.000 cells/well in 100 µl medium. After one hour wells were washed twice with PBS and adherent cells were detected using crystal violet assay. To analyze phagocytosis confluent ARPE–19 cells were incubated for 3 hours at 37° C with SNAFL–2 fluorescent dye labeled ROS, washed with media to remove non–phagocytized ROS, trypsinized, centrifuged, resuspended (10⁶ cells per tube) in flow cytometry buffer and 20.000 cells were analyzed with a flow cytometer. Tight junctions were evaluated by measuring TER of confluent cells cultured on 0.02µm pore size inserts coated with collagen type IV, fibronectin and laminin. Results: Attachment on fibronectin–coated wells and laminin–coated wells was approximately twice as high as attachment on plastic wells. Attachment on collagen–coated wells was significantly higher than on plastic, but substantially less than attachment on fibronectin or laminin coated wells. The phagocytic activity of RPE cells was highest on fibronectin–coated substratums (SFI = 3.74 ± 0.39) followed by laminin (SFI = 3.45 ± 0.38), and collagen IV (SFI = 3.33 ± 0.83) when compared to uncoated dishes (SFI = 2.87 ± 0.25). However, only the difference between non–coated substratum and fibronectin–coated substratum is statistically significant. TER measurements did not appear to be significantly different on coated filters than on control, non–coated filters; however, more studies need to be done to determine morphologically the state of monolayers and to correlate the presence of junctional molecules to TER. Conclusions: These studies show that extracellular matrix components do modulate cell attachment and phagocytic activity.

Keywords: retinal pigment epithelium • retinal culture • protein structure/function