Time at confluence for human RPE cells: effects on the adherens junction and in vitro wound closure.

PURPOSE: To determine how time at confluence affects the properties of cultured human retinal pigment epithelium (RPE) cells, with emphasis on the adherens junction. METHODS: Cultures were maintained at confluence without passage for intervals to several months. Adherens junction proteins (N-cadherin, E-cadherin, alpha-catenin, beta-catenin, plakoglobin, and actin) and the proliferation marker Ki-67 were localized in the cultures by fluorescence microscopy, and in vitro wound healing was compared. Adherens junctions were analyzed for protein solubility in detergent buffers and sensitivity to disruption by treatment with anti-cadherin antibodies and low calcium conditions. RESULTS: Compared with cultures in early-confluence (2-3 days), postconfluent cultures (weeks) had more mature adherens junctions characterized by a circumferential (rather than linear) actin organization, and a zonular (rather than punctate) distribution of more detergent resistant cadherin and catenins. Postconfluent cultures also had fewer Ki-67-positive cells and a higher cell packing density. Early-confluence cells migrated into in vitro wounds as dissociated single cells, whereas postconfluent cells moved as contiguous sheets, retaining an intact junction during wound-induced cell migration and proliferation. Mature junctions were not disrupted by treatment of living cells with N-cadherin antibodies, which bound to and remained detectable at junctions for several days. Calcium withdrawal displaced N-cadherin from mature junctions and rendered it more soluble, but the dominant circumferential pattern of actin was stable. Restoration of medium calcium resulted in a rapid (hours) recovery of a nearly complete zonular pattern of insoluble N-cadherin. CONCLUSIONS: Over long postconfluent periods, cultured RPE cells became more growth quiescent, and intercellular cadherin adhesions became more stable, exhibiting increased resistance to calcium removal and greater retention of junctional integrity during in vitro wound closure. Consideration should be given to whether the behavior of RPE cells in postconfluent cultures, where intercellular adhesions are more mature, more closely simulates RPE cells in situ than cells in early-confluence cultures, which are more commonly used for analysis.

Expression of E-cadherin by human retinal pigment epithelium: delayed expression in vitro.

PURPOSE: To determine whether retinal pigment epithelial (RPE) cells, which reportedly express N-cadherin as their major cadherin cell adhesion protein, also express the more common epithelial cadherin, E-cadherin. METHODS: Cadherins expressed by human RPE cells in situ were examined by western blot analysis of extracts prepared from the RPE of human adult eyes. Cadherins expressed in vitro were examined by analysis of confluent and postconfluent human RPE cultures, using the methods of reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Protein distribution was examined by conventional fluorescence microscopy, confocal imaging, or both. Proteins whose expression, distribution, or both correlated with E-cadherin expression in other epithelial cells were examined by similar methods in cultured RPE cells. RESULTS: In addition to N-cadherin, E-cadherin (and P-cadherin) was found in adult human RPE in situ. In cultured human RPE cells, N-cadherin was ubiquitous, but E-cadherin was limited to patches of cells and was not expressed until several weeks after confluence, a time when several phenotypic variants become prominent. E-cadherin was absent from RPE cells of fusiform shape but was found in only a subset of epithelioid RPE cells. Unlike epithelial cell lines expressing E-cadherin, cultured RPE cells with E-cadherin did not show diminished coexpression of N-cadherin, increased expression of desmosomal proteins, or a preferential expression of the alphaE- (rather than alpha-N) isoform of the cadherin linker protein alpha-catenin. Na/K ATPase distributed to both apical and basolateral membranes in RPE cells with junctional E-cadherin and not preferentially to the basolateral domain as in most epithelial cells with E-cadherin. CONCLUSIONS: RPE cells express E-cadherin, a cadherin found in most other epithelial cells, but which was believed to be absent from RPE. In RPE in vitro, E-cadherin expression is a late developmental event, occurring in late confluence in cells that already express N-cadherin. E-cadherin is an established epithelial morphoregulatory protein, but it does not induce the same properties in RPE cells as in other epithelial cells, suggesting tissue-specific differences in the potential of E-cadherin to determine an epithelial phenotype.

Autofluorescent inclusions in long-term postconfluent cultures of retinal pigment epithelium.

PURPOSE: To examine the accumulation and morphologic features of fluorescent inclusions in retinal pigment epithelial (RPE) cells in vitro and to determine whether accumulation correlates with parameters of age. METHODS: Cultured human RPE cells were maintained undisturbed at confluence for intervals as long as 2 years and then were examined for autofluorescent inclusions by fluorescence and electron microscopy, with comparisons to the lipofuscin of freshly isolated RPE. Autofluorescence was also examined in bovine RPE cultures that were aged in vitro by replicative senescence. In some postconfluent cultures,the activity of the lysosomal enzyme cathepsin D was measured, and the effect of cell growth on autofluorescence was examined by reinducing cell proliferation in late-stage cultures. RESULTS: Autofluorescent inclusions accumulated in human and bovine RPE cultures after extended postconfluent periods. Accumulation was not accelerated in cultures from older donors or in cultures that were aged in vitro. The number of granules per RPE cell varied for lipofuscin in situ and inclusions in vitro, although the latter were more heterogeneous in size and shape and in ultrastructural appearance of the granule contents. Lipofuscin and autofluorescent inclusions were lost when RPE cells were propagated, but in contrast to lipofuscin, the inclusions became smaller and fainter when growth was reinduced in postconfluent cultures. Cathepsin D activity varied among cultures and showed no significant change as time elapsed after confluence. Activity increased after repropagation of long-term postconfluent cultures. CONCLUSIONS: In the absence of phagocytic challenge with photoreceptor outer segments, postconfluent RPE cultures accumulate heterogeneous materials that show autofluorescence. Accumulation is associated with one marker of age: time at confluence, which simulates a long-term, nonmitotic state similar to that which occurs in situ during aging in postproliferative tissues. The autofluorescent inclusions in vitro predictably differ from autofluorescent lipofuscin in situ, presumably because the inclusions formed in culture are derived from RPE autophagy alone. Postconfluent cultures can be used to examine the molecular and biologic properties of materials originating only from an autophagic source, or coupled with phagocytic challenge to produce a model of developing RPE lipofuscin that has an autophagocytic and a heterophagic component, similar to RPE cells in situ.

Cell-cell adhesion molecules and the development of an epithelial phenotype in cultured human retinal pigment epithelial cells.

For most epithelial cells, the adherens junction protein E-cadherin is an epithelial morphogen, inducing the development of an epithelial phenotype in vitro after cell contact at confluency. Here retinal pigment epithelial cells (RPE), which lack E-cadherin but express a cadherin that is also found in many non-epithelial cells (N-cadherin), were examined for the ability to produce an epithelial phenotype in vitro. Subpopulations of grossly epithelioid or fusiform cells were selected for analysis from RPE cultures derived from adult human donors. After confluency, epithelioid RPE cells were observed to undergo time-dependent changes that were similar to those previously found in epithelial cells expressing E-cadherin: the cadherin gradually developed a zonular distribution of detergent-resistant protein that co-localized with forming circumferential actin bundles; Na/K ATPase accumulated at cell contact sites, then polarized to its tissue-specific domain (the apical membrane for RPE); the cells formed elevated domes on the impermeant culture substrate. In contrast to cells expressing E-cadherin, these events in RPE required weeks rater than days at confluency. Additional proteins were examined in epithelioid RPE cells revealing that cytokeratins reorganized after confluency producing a zonular array, and several other adhesion proteins (alpha5beta1 integrin, ICAM-1, PECAM-1, NCAM) became enriched at cell-cell contact sites, each developing a distinct pattern at a distinct postconfluency interval. In contrast to epithelioid RPE, in fusiform RPE the adhesion molecules did not develop discrete distribution patterns after confluency, although the same complement of adhesion proteins was expressed. In cells expressing E-cadherin, the absence of epithelial properties is often due to underexpression of the cadherin or of the catenins, adherens junction proteins that link the cadherin to actin. Fusiform RPE, however, were not deficient in these proteins, expressing amounts of N-cadherin, alpha-catenin, beta-catenin, plakoglobin, p120, alpha-actinin and vinculin that were equivalent to epithelioid cells. It appears, therefore, that a subset of epithelial cells that express N-cadherin can produce a highly-developed epithelial phenotype in vitro through a slow morphogenetic process. However, the expression alone of adhesion molecules, including those with a morphoregulatory function in other cells, is insufficient to produce an epithelial phenotype in all cells derived from the pigment epithelium.

Phenotypic heterogeneity of retinal pigment epithelial cells in vitro and in situ.

We have previously developed methods based upon differential cell adhesion to select for cells of two different phenotypes, epithelioid and fusiform, from cultures of human RPE. Here we considered whether the differences in cell shape correlated with differences in protein tyrosine phosphorylation since it is known that elevated phosphorylation perturbs the stability of the adherens junction, a major determinant of epithelial phenotype. In cultures of both phenotypes we found low tyrosine phosphorylation levels in postconfluent cultures, and the same complement of tyrosine phosphoproteins after treatment with a phosphatase inhibitor. However, in contrast to epithelioid cells, fusiform RPE failed to localize the phosphoproteins to junctional sites on the cell periphery. We also re-evaluated primary and passaged RPE cultures for additional cell shape variants. Several discrete phenotypes were identified within the same cultures. They required several weeks at confluency to develop in primary as well as in passaged cultures, but after developing they remained stable for months. Since explanted RPE cells manifest several shape variants in an identical culture environment we examined bovine RPE cells in situ to determine whether the cells were heterogenous with regards to some cell surface and cytoskeletal proteins that might contribute to cell shape. Circumferential actin microfilament bundles and the occluding junction protein ZO-1 had fairly uniform distributions among cells in the monolayer, but the intermediate filament protein vimentin and the pericellular expression of phosphotyrosine varied among individual cells. Therefore, despite its grossly homogeneous appearance, the RPE monolayer in situ might be considered a mosaic of structurally heterogeneous cells which can give rise to phenotypically-distinct subpopulations when propagated in vitro.