Isolation of a retinal pigment epithelial cell-derived fraction which promotes Müller cell proliferation.

Retinal pigment epithelial cells (RPE) secrete a factor(s) in vitro which promotes Müller cell proliferation. To begin the isolation of the active factor, medium conditioned by cultured RPE (RPE-CM) was fractionated by fast protein liquid chromatography. The fractions were tested in an assay for Müller cell proliferation and it was found that one fraction contained biological activity comparable to that of complete RPE-CM. Gel electrophoresis demonstrated that this fraction consisted of proteins with approximate molecular masses of 35-88 kDa. An affinity assay revealed that only an 88-kDa protein in the RPE-CM binds to Müller cells. This protein was also present in the active fraction; therefore, it is the most likely source of the mitogenic activity of RPE-CM.

Retina-derived fetuin (RDF): analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot.

PURPOSE: This study was undertaken to determine the presence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light micros-copy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. METHODS: Retinal tissues and sections from embryonic, early postnatal and adult normal rats and retinal pigment epithe-lial (RPE) cells from postnatal rats were immunostained for fetuin with a polyclonal fetuin antibody and a peroxidase conjugated-secondary antibody using immunocytochemistry and Western blot analyses. The cDNA generated from RNA isolated from early postnatal rat retinas and RPE was probed with primers for rat fetuin, amplified by PCR and the PCR products were analyzed by Southern blot. RESULTS: Fetuin (RDF) was immunolocalized to cells of the neuroepithelium in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, inner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve processes were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicating that this protein may be developmentally regulated. Using RT-PCR, the message for rat fetuin was demonstrated in the retina and RPE of normal postnatal rats. Southern blot confirmed that the PCR product from the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin. CONCLUSIONS: Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cysteine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.

Müller and retinal pigment epithelial (RPE) cell expression of NGFI-A and c-fos mRNA in response to medium conditioned by the RPE.

Retinal pigment epithelial (RPE) cells secrete a factor(s) which promotes Müller and RPE cell survival and proliferation in vitro. These influences may play developmental and functional roles as well as contribute to ocular pathologies such as proliferative vitreoretinopathy (PVR). In the past few years, a number of immediate early genes (IEGs) have been identified. Many IBGs encode transcription factors, the expression of which is altered by stimuli such as growth factors. Since an RPE-derived factor(s) elicits proliferation of Müller and RPE cells, we investigated the expression of two IEGs, NGFI-A and c-fos, in both cell types after treatment with medium conditioned by the RPE (RPE-CM). We found that Müller and RPE cells had increased levels of NGFI-A mRNA following treatment with RPE-CM; in contrast, only a slight increase in c-fos mRNA was induced in RPE, but not Müller cells. Immunolabeling for NGFI-A protein revealed nuclear staining in both cell types which corresponded with the increased mRNA levels in RPE-CM-treated cultures. This in vitro study demonstrates a potential mechanism by which RPE-secreted factors may exert autocrine or paracrine effects on retinal cells in vivo. Specifically, NGFI-A may be the primary target of a second messenger system that is regulated by an RPE-derived factor(s).

Müller cell survival and proliferation in response to medium conditioned by the retinal pigment epithelium.

Müller cells have been implicated in the pathogenesis of proliferative vitreoretinopathy and subretinal scar formation; however, the source(s) and signal(s) responsible for their activation are unknown. This study was undertaken to determine if the retinal pigment epithelium (RPE) could be involved in this signaling process by studying its effects on Müller cell survival and division in vitro. A pure population of Müller cells isolated from 1-2 day Long-Evans rats was seeded at low density and treated with medium conditioned by neonatal rat RPE (RPE-CM) or a nonconditioned, defined medium. By day 3, Müller cells cultured in RPE-CM increased in number 2-fold. These cells survived up to 21 days, which was the longest time tested. In contrast, cell number decreased in control wells 75% by day 3, and 100% by day 4. The RPE-mediated survival and proliferation of the Müller cells occurred in a dose-dependent manner. The mitogenic response was specific for the RPE when compared with fibroblasts and non-retinal epithelial cells. Heat and trypsin treatment of the RPE-CM completely abolished its survival and mitogenic activity. These findings demonstrate the establishment of an in vitro model which can be used to investigate RPE-Müller cell interactions. This study also provides evidence for RPE involvement in Müller cell interactions. This study also provides evidence for RPE involvement in Müller cell survival and proliferation.