Effects of extracellular matrix and Bruch's membrane on retinal outer segment phagocytosis by cultured human retinal pigment epithelium

Abstract

The goal of this study was to determine the effect of extracellular matrix components on the phagocytic function of the retinal pigment epithelial (RPE) cell. Cultured human fetal RPE cells were established in culture and plated on three commercially-prepared substrates: collagen IV, fibronectin and laminin and on three native matrices: bovine corneal endothelial cell matrix (BCEM) denuded bovine Bruch's membrane and denuded human Bruch's membrane. Cultured cells were allowed to become confluent and maintained for an additional two weeks before uptake of fluorescent bovine retinal outer segments (ROS) was measured by flow cytometry. Morphology by phase contrast microscopy and melanization was also determined as measures of differentiation. The results showed that morphology, melanization and ROS uptake by cells on collagen IV, laminin and fibronectin were not different from control cells plated on tissue culture plastic. However, ROS uptake by cells plated on BCEM was significantly less than that of cells cultured on plastic and melanization was greater. ROS uptake by cells plated on both types of Bruch's membrane was also significantly less than control cells. Treatment of cells plated on tissue culture plastic with 44 mM NaHCO3, which increased melanization, also reduced ROS uptake. We conclude that native matrices seem to contain components that significantly depress ROS uptake in culture. The inhibition is not mimicked by collagen, laminin or fibronectin coated wells. The ECM may play a significant role in controlling phagocytosis of ROS either by determining morphology, increasing differentiation or by directly influencing intracellular metabolism, and thus serve as another level of control for this RPE function which may not occur in cells plated on tissue culture plastic. These results may also have implications for the effects of aging or disease in which there are changes in the ECM.

Loss of RPE phenotype affects phagocytic function

“…The present study demonstrates that immunolabeling of αvβ5 integrin also redistributes dramatically from apical patches on the normal RPE microvillous surface to a dispersed and diminished distribution on the vitreous-transformed RPE, which corresponds to a significant reduction in the expression of αvβ5 integrin and a decrease in phagocytic ability.In general, cultured RPE cells show heterogeneity and can be classified into several groups based on their morphology [27,28]. Several investigators have observed that less differentiated or fusiform-shaped RPE cells actually phagocytize more ROS than well-differentiated polygonal and pigmented cells [29,30,31]. The transformed cultures induced by vitreous in our study have a morphology similar to the fusiform cells described in the above studies; however, their phagocytic activity is significantly decreased.…”

Nitrite-Modified Extracellular Matrix Proteins Deleteriously Affect Retinal Pigment Epithelial Cell Function and Viability: A Comparison Study with Nonenzymatic Glycation Mechanisms

“…It has been shown that the ECM impacts upon the phagocytic capability of RPE. 59 Although not specifically examined in this study, it can be hypothesized that matrix modification, owing to its deleterious effects on RPE cell viability, could impair the processing of photoreceptor derived compounds such as A2E and contribute to the accumulation of intracellular lipofuscin. This would again create a damaging cycle in which matrix modification results in increased A2E accumulation, which imparts increased toxicity to cells located on damaged, modified ECM proteins.…”

Collagen XVII and BPAG1 expression in the retina: Evidence for an anchoring complex in the central nervous system

“…The rod photoreceptor outer segment is an elongated membrane-enclosed stack of membranous discs, whereas the cone outer segment is a single continuous, but invaginating, membrane. Outer segments and RPE interaction via the interphotoreceptor matrix molecules are essential to prevent retinal detachment and for the constant renewal of the outer segments (Lazarus and Hageman, 1992;Miceli and Newsome, 1996). In rods, there is considerable turnover of the outer segments, with new discs generated by invaginations of the membrane at the base of the outer segment and older disc material Fig.…”