Role of Protein Interactions in Retina Development and F

National Eye Institute

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Abstract

Work in this research group is aimed at elucidating the mechanisms that regulate the activities of pigment epithelium-derived factor (PEDF), an extracellular serpin with neurotrophic and antiangiogenic activities. We investigated the distribution of PEDF protein in the monkey retina. The concentration of this soluble factor was found greater in the interphotoreceptor matrix than in vitreous and aqueous. PEDF was detected at significant levels surrounding rod and cone outer segments, and at lower levels in the retinal pigment epithelium (RPE) as visualized by confocal microscopy. PEDF transcripts were highly expressed in RPE as compared with several other monkey ocular tissues by northern blotting. We established primary cultures of monkey RPE cells that highly expressed PEDF. When polarized on permeable supports these cells secreted PEDF preferentially to the apical side, i.e., the interphotoreceptor matrix in the native retina. We investigated the effects of extracellular matrix metalloproteinases (MMPs) on PEDF. Limited proteolysis with MMP-2 and MMP-9 degraded PEDF in a Ca+2-dependent fashion to yield undetectable products that lacked neuroprotective and antiangiogenic properties. The effects of hypoxia on PEDF degradation were examined. In vivo PEDF decreased in the RPE/choroid when tissue hypoxia was induced in the Retinopathy of Prematurity mouse model. In cultured monkey RPE cells, exposure to low oxygen or to chemical hypoxia mimetics decreased PEDF levels in the media, with no significant change for PEDF mRNA, but with increases in MMP-2 activities that degraded PEDF in a dose-dependent fashion. The effects of VEGF on PEDF proteolysis were explored. BHK cells produced MMP-2 activities that degraded PEDF with identical protease inhibition pattern as those in vitreous. VEGF promoted increases in MMP-2-mediated PEDF proteolysis in a concentration dependent fashion. We examined the neurotrophic effects of PEDF and erythropoietin (Epo) on immortalized retinal precursor cell lines. PEDF and Epo increased the viability of serum-deprived and depolarized retinal cells in a dose-dependent fashion, Epo being more potent than PEDF. PEDF specifically bound to receptors on the surface of the retinal cells, but did not bind to the soluble Epo receptor in real-time or promoted erythroid differentiation. We worked on the isolation, cloning, expression and characterization of a putative receptor for PEDF. A yeast 2-hybrid system identified a PEDF interactant gene with similarity to an unknown liver orphan receptor. Sequence alignments of the interactant with DNA databases identified a novel gene from RPE, named PEDF-R1. Experiments in silico, in vitro and in cell culture demonstrated that its gene product was a transmembrane protein with binding affinity for PEDF. We studied the structure-function relationships of PEDF?s antiangiogenic activity. We compared the efficacy of PEDF peptides, other antiangiogenic factors and serpins using an ex vivo chick aortic arch assay. PEDF and 34-mer, a small PEDF peptide towards the amino-end region, inhibited vessel sprouting, being PEDF and 34-mer >10-fold more efficient than 44-mer, a peptide region contiguous to the 34-mer, and ovalbumin, but less efficient than angiostatin., Peptide 34-mer retained the antiangiogenic activity of PEDF on HUVEC cell migration assays and interacted with identical signal transduction pathways as did the full length PEDF. Both PEDF and 34-mer bound to cell surface receptors on HUVECs. We developed a new method for evaluating and quantifying chroroidal neovascularization (CNV) in rodents. CNV lesions were induced with laser and the morphology and volume of the lesions evaluated in choroid/RPE flat mounts by confocal microscopy using 3-D reconstructions. This method allowed us to evaluate early changes after laser-induced CNV. We investigated the in vitro and in vivo transcleral-RPE permeability of PEDF and ovalbumin. Confocal microscopy, transepithelial resistance and impermeability to trypan blue and horseradish-peroxidase confirmed confluency, polarization and tight junction formation of RPE cell monolayers cultured on permeable supports. Fl-PEDF and Fl-ovalbumin passed through polarized RPE cells from either the apical or basal side. Fl-ovalbumin diffused through the scleral tissue at a constant rate. In rat eyes, subconjunctival Fl-PEDF injections or Fl-ovalbumin implants revealed diffusion into the retina as early as 1 hour, with maximum diffusion by 24 hours.