We completed studies on a novel inhibitor of 5-lipoxygenase (5-LOX) that prevents oxidative stress-induced cell death of RPE cells. 5-LOX oxygenates arachidonic acid to form 5-hydroperoxyeicosatetraenoic acid, which is further converted into biologically detrimental leukotrienes, such as leukotriene B4 (LTB4). We investigated the role of PEDF-R on the 5-LOX pathway in oxidative stress of RPE. LOX activity assays were performed with soybean and potato LOX. Binding was evaluated by peptide-affinity chromatography and pull-down assays with PEDF-Rderived synthetic peptides or recombinant protein. Oxidative stress was induced in human ARPE-19 and primary pig RPE cells with H2O2/TNF-. Reverse transcriptionPCR of ALOX5 and PNPLA2 genes was performed. PEDF-R and 5-LOX levels were assayed by immunoblotting. Reactive oxygen species (ROS) levels, cell viability and death rates were determined. Leukotriene B4 levels were measured by ELISA. Among five peptides spanning between positions Leu159 and Met325 of human PEDFR polypeptide, only two overlapping peptides, E5b and P1, bound and inhibited LOX activity. Human recombinant 5-LOX bound specifically to peptide P1 and to His6/Xpress-tagged PEDF-R via ionic interactions. Peptides E5b and P1 promoted viability and decreased death of RPE cells undergoing oxidative stress. P1 decreased the generation of ROS in ARPE-19 cells in a dose-dependent fashion. Oxidative stress decreased the levels of PNPLA2 transcripts and PEDF-R protein with no effect on ALOX5 expression and 5-LOX protein, showing PEDF-R regulation with oxidative stress. P1 additions or overexpression of the PNPLA2 gene decreased both LTB4 levels and RPE cell death undergoing oxidative stress. Thus a novel peptide region of PEDF-R inhibits 5-LOX, which intersects with RPE cell death pathways induced by oxidative stress. We performed RT-PCR to evaluate expression of Keap-1 and Nrf-2 genes, shown to be part of relevant pathways for oxidative stress. To determine the polarization status of ARPE-19 cells in tissueculture treated plastic, confluent cells were labelled with Ezrin (an apical marker for RPE) and ZO-1 (marker of tight junctions) and DAPI to stain the nuclei. The cells were examined by confocal microscopy (NEI BioImaging core, in consultation with Dr. Bob Fariss). The XZ plane showed staining patterns characteristic of polarized RPE. We continued studies on the serpin protease nexin-1 (PN-1), encoded by the SERPINE2 gene that has neurotrophic effects in the brain and inhibits angiogenesis in the retina. We investigated its efficacy in cell survival and compared it to PEDF. Recombinant human PN-1, a version of PN-1 lacking serine protease inhibitory activity referred to as R346A PN-1, PEDF proteins, and synthetic 17mer peptides were used. Survival activity in serum-starved rat-derived retinal precursor (R28) cells was assessed with TUNEL cell death assays with or without a selective and competitive catalytic inhibitor of PEDF-R. Serum-starvation resulted in a decrease of Bcl-2 levels leading to R28 cell death. The effect of PN-1, R346A PN-1 and PN-1 17mer on Bcl-2 levels were tested. Given that Sirtuin-1 levels decrease with retinal degenerations and aging, we evaluated the effect of these molecules on Sirtuin-1 transcript levels. Cell death occurring during serum-starvation of R28 cells was followed with commercial kits for the development of high throughput assays by measuring DNA fragmentation. The effect of PEDF on oxidative stress induced cell death on iPSC-RPE cells was investigated. An assay to test optimal conditions for oxidative stress by different compounds, such as, paraquat and sodium iodate was set up. At endpoint, cytotoxicity was measured with LDH and viability was measured with cell TiterGlo. The effect of PEDF and PN-1 to protect these cells was evaluated. We developed degenerative models in vitro using ARPE-19 cells and cone-type photoreceptors 661W cells exposed to H2O2 and sodium iodate. Characterization of the degenerative course was performed using cytotoxicity and cell viability assays. The concentration-dependent effects of the toxic agents were assayed. The protective effects of PEDF, PN-1 R346A PN-1 proteins, and 44mer and 17mer peptides were tested against oxidative stress agents in the cells. The survival effects of PEDF and its peptides in retinal photoreceptors during development in vitro were investigated (collaboration with Dr. Politi). The blocking effects of P1, a PEDF receptor blocking peptide were assayed. The neuroprotective effects of PEDF and PEDF peptides infocal light-emission-diode-induced phototoxicity in adult albino mice were also explored (collaboration with Dr. Vidal-Sanz). The effects of ionophore A23187 and phosphodiesterase inhibitors Zaprinast and IBMX on cone-type 661W cells viability, cytotoxicity, cGMP levels and calcium accumulation were assayed. Pnpla2 transcripts and PEDF-R protein levels were determined in cells treated with these molecules. The cytoprotective effects of PEDF and PEDF peptides with and without PEDF-R inhibitors or blocking peptides vs. these compounds were evaluated. To characterize the PEDF-R activity that is crucial for the PEDF-mediated retina cell survival, we began to investigate the lipid composition of mouse retinas by Imaging Mass Spectrometry using the state of the art technology of matrix assisted laser desorption imaging mass spectrometry (MALDI-IMS), for direct analysis of the classes of lipid molecules associated with PEDF-R activity in the retina. Expression vectors for untagged full-length human PEDF versions with single alterations at R99A and H105A, and lacking the ability to bind heparin, collagen, or hyaluronan were constructed using the pCEP4 vector designed for protein expression in HEK293 Ebna cells. Recombinant human PEDF protein was purified from media of baby hamster kidney cells harboring expression vectors pMA-PEDF by two-step ion-exchange column chromatography. Isoforms of the protein were separated, dialyzed against PBS and stored for further use in biological, biochemical and biophysical assays. Expression vectors for full length-human PEDF-R versions with an N-terminal Xpress/6XHis tag and lacking the C-terminal end and binding site for PEDF were transformed into BL21 bacterial cells, and the recombinant proteins expressed as insoluble inclusion bodies and refolded. A purification protocol for recombinant PEDF-R versions was optimized. The recombinant proteins were analyzed by SDS-PAGE and western blots, and tested for binding to fluoresceinated PEDF and their phospholipase and triglyceride lipase activities assayed under various buffer conditions. A high-throughput screen was optimized to identify regulators of lipase activity for PEDF-R. We began studies on CGI-58 and G0S2, known regulators of the PEDF-R triglyceride lipase activity. To determine if the proteins regulate the PEDF-R phospholipase activity, expression vectors for full-length human CGI-58 with a C-terminal myc-flag tag were transfected into COS-7 cells, and cell lysates analyzed for protein expression by immunoblotting. P1 binding assays were performed with lysates. CGI-58 lysates were tested for cytoprotective activity in cell cultures. Bacterial expression vectors containing an N-terminal 6XHis tag and full-length human CGI-58 or G0S2 were constructed and transformed into BL21 bacterial cells. Expression and purification protocols were optimized to produce purified soluble recombinant CGI-58 and G0S2 proteins. We continued the collaborative study investigating the role of PEDF in type V OI with clinical characteristics of type VI OI. PEDF levels were determined in media of ARPE-cells under serum starvation, exposed to PAM, and treated with osteo-inducers that produce BRIL. The topology of BRIL in ARPE-19 was also investigated by trypsin digestion.
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