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Cultured pigment epithelial cells of the fetal human retina secrete a protein, pigment epithelium-derived factor (PEDF), that induces a neuronal phenotype in cultured human retinoblastoma cells. Morphological changes include the induction of an extensive neurite meshwork and the establishment of corona-like cellular aggregates surrounding a central lumen. The differentiated cells also show increases in the expression of neuron-specific enolase and the 200-kDa neurofilament subunit. Amino acid and DNA sequence data demonstrate that PEDF belongs to the serine protease inhibitor (serpin) family. The PEDF gene contains a typical signalpeptide sequence, initiator methionine codon, and polyadenylylation signal and matches the size of other members of the serpin superfamily (e.g., a1-antitrypsin). It lacks homology, however, at the putative serpin reactive center. Thus, PEDF could exert a paracrine effect in the embryonic retina, influencing neuronal differentiation by a mechanism that does not involve classic inhibition of serine protease activity.The retinal pigment epithelium (RPE) develops in advance of and lies adjacent to the neural retina. A closed compartment between the two cell layers contains the interphotoreceptor matrix and many soluble secretory products of RPE and neural retinal cells. In the human, the interphotoreceptor matrix is present by 20 weeks of gestation and contains at least one specific marker, the interphotoreceptor retinoidbinding protein (1). Nutrients, metabolites, or trophic factors exchanged between the RPE and neural retina must pass through the interphotoreceptor matrix. RPE cells, for example, are thought to synthesize and secrete a photoreceptor survival-promoting factor, PSPA (2). Cultured RPE cells also synthesize a number of other, well-known trophic factors including PDGF (3), FGF (4), TGF-a (5), and TGF-13 (6). It is thus possible that these or other, unknown factors derived from the RPE could influence neural retina development.We Cell Culture. The Y79 retinoblastoma cell line (American Type Culture Collection) was maintained in suspension culture (7). Cells were harvested by centrifugation for 5 min at 600 x g, washed in prewarmed serum-free medium, centrifuged again, and suspended at 106 cells per ml in serum-free medium with or without HPLC-purified PEDF (50-500 ng/ ml) (9) for a 7-day incubation period prior to attachment to poly(D-lysine)-coated flasks (8). A number of growth factors were tested under similar conditions: NGF at 10-300 ng/ml, PDGF at 1-5 ng/ml, TGF-a at 5 ng/ml, TGF-f3 at 5 ng/ml, FGF at 25-220 ng/ml, and EGF at 25-100 ng/ml (11).Cell Analyses.

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We investigated gene expression profiles of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in differentiated and non-differentiated retinal pigment epithelial (RPE) cells during oxidative stress. Human RPE cells were grown in culture on laminin-coated flasks to obtain differentiated features. Cells cultured on plastic were used as non-differentiated controls. After confluence, hydrogen peroxide (H2O2) was added for 48 h, then, total RNA was extracted and used for RT-PCR and Northern blot analysis. Medium conditioned by RPE was used for ELISA, Western blotting, and in vitro angiogenesis assay. As a result, differentiated RPE cells expressed significantly higher levels of VEGF protein, as compared to their non-differentiated counterparts. The expression pattern remained consistent even after cellular exposure to H2O2. Conversely, while elevated levels of PEDF transcript and protein were seen in differentiated RPE cells, compared to non-differentiated cells, a marked decrease at both PEDF mRNA and protein levels was seen after treatment with H2O2. Moreover, this decrease in PEDF expression was dosage dependent. In in vitro angiogenesis assay, conditioned medium from differentiated human RPE cells after exposure to H2O2 showed a dramatic increase in tubular formation and migratory activity of microvascular endothelial cells. These data suggest that, in physiological conditions, a critical balance between PEDF and VEGF exists, and PEDF may counteract the angiogenic potential of VEGF. Under oxidative stress, PEDF decreases disrupting this balance. This equilibrium shift may be significant in promoting a pathological condition of RPE cells and contributing to choroidal neovascularization in age-related macular degeneration.

Retinal pigment epithelial (RPE) cells form a functional complex with photoreceptor neurons of the retina, interacting through the interphotoreceptor matrix (IPM). We now provide evidence that the gene for pigment epithelium-derived factor (PEDF), a protein possessing neurotrophic and neuronal-survival activities, is highly expressed by both fetal and young adult RPE cells. PEDF mRNA is present in RPE cells of the human eye at 17 weeks of gestation, demonstrating its potential for action in vivo during early retinal development. The PEDF protein is secreted in vivo where it constitutes a part of the fetal and adult IPM surrounding photoreceptor outer segments. A polyclonal PEDF antibody recognizes at least four isoforms of secreted human and bovine PEDF by two dimensional gel analysis, and detects a similar 50 kDa protein in the IPM of several other vertebrate species. Within soluble extracts of RPE cells, however, where little, if any, of the 50 kDa species can be detected, an immunoreactive 36 kDa protein is observed by Western blot analysis. By immunofluorescence, PEDF is localized intracellularly in association with the nucleus, presumptive secretory granules, and cytoskeletal elements of cultured RPE cells with PEDF and actin antibodies colocalizing to the same cytoskeletal structures. During initial stages of attachment, PEDF and actin also concentrate at the tips of pseudopods extended by the cultured RPE cells. However, with successive passages, synthesis, and secretion of the PEDF protein as well as transcription of its mRNA decrease and are lost by about 10 passages. In parallel, cultured RPE cells lose their proliferative potential and change from an epithelial-like morphology in early passages to a more fibroblast-like appearance by about the 10th passage. PEDF is thus apparently present intracellularly and extracellularly in both fetal and early adult periods where it could be involved in cellular differentiation and survival and with its loss, in the onset of senescence.

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The authors propose that a mitochondrial complex I defect is associated with the degeneration of TM cells in patients with POAG, and antioxidants and MPT inhibitors can reduce the progression of this condition.