Work in this research group is aimed at elucidating the mechanisms that regulate the activites of pigment epithelium-derived factor (PEDF), an extracellular neurotrophic and antiangiogenic protein. PEDF induces neuronal differentiation on retinoblastoma Y-79 cells via interactions with cell-surface receptors in these cells. It also has binding affinity for glycosaminoglycans. We investigated the effects of glycosaminoglycans on PEDF-receptor interactions. Using specific glycosaminoglycan degrading enzymes in spectrophotometric assays and PEDF-affinity chromatography, we detected heparin and heparan sulfate-like glycosaminoglycans in the Y-79 conditioned media, which had binding affinity for PEDF. The Y-79 conditioned media significantly enhanced the binding of 125I-PEDF to Y-79 cell-surface receptors. However, enzymatic and chemical depletion of sulfated glycosaminoglycans from the Y-79 cell cultures by heparitinase and chlorate treatments decreased the degree of 125I-PEDF binding to cell-surface receptors. Thus heparin/heparan sulfate may be important in regulating the activity of PEDF by providing efficient cell-surface receptor binding. To investigate the effects of hypoxia on PEDF, we exposed retina pigment epithelial (RPE) cells to low oxygen or to chemical agents that mimic hypoxia. Hypoxia reduced the PEDF levels in the RPE media, but its gene expression remained unchanged compared to normoxic controls. Hypoxia induced gelatinolytic activities in the RPE media, which were EDTA-sensitive and calcium-dependent, degraded PEDF, and immunoreacted with MMP-2 antibodies. Vitreous and BHK[pPEDF] cell conditioned media also contained these activities. Purified MMP-2 and MMP-2 induced in BHK[pPEDF] cells by VEGF were able to specifically and completely degrade PEDF. Thus hypoxia can downregulate PEDF expression at a post-translational level by MMP-2 proteolytic degradation. We also investigated the effects of dexamethasone on PEDF in collaboration with the laboratory of Dr. T. Borras (UNC). Dexamethasone was used to perfuse anterior segment organ cultures prepared from paired eyes from post-mortem human donors and treat cultured cells from human trabecular meshwork (HTM). Dexamethasone increased the PEDF mRNA of trabecular meshwork from perfused segments over contralateral controls and of cultured HTM cells over vehicle-treated cells. It also increased the levels of secreted PEDF protein in effluents of organ cultures and in HTM cell media above controls. Confocal microscopy of HTM tissue showed a positive correlation between increased PEDF immunofluorescence and dexamethasone-perfusion. Thus dexamethasone can upregulate PEDF expression at a transcriptional level. To establish novel routes of protein delivery to the retina, we evaluated the diffusion of fluorescein-conjugated proteins, PEDF (50-kDa) and ovalbumin (45-kDa), through monolayers of monkey RPE cells and pig sclera tissue explants, and in rat eyes in vivo. Transepithelial resistance and voltage, as well as the lack of trypan blue and horseradish peroxidase diffusion confirmed confluency and tight junction formation of the cultured RPE cells. Fl-PEDF passed through the RPE monolayers from either the apical or basal side, as determined by immunoblotting, fluorometry, and confocal microscopy. Fl-ovalbumin diffused through the scleral tissue at a constant ratio, as determined by fluorometry (in collaboration with the laboratory of R. Lutz, OD/ORS). In animal models injected subconjunctivally with Fl-PEDF or with Fl-ovalbumin implants, microscopy revealed fluorescein diffusion into the choroid/RPE complex and into the photoreceptor outer segments suggesting that subconjunctival protein delivery can be a means for increasing the PEDF levels in the retina.
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