The protective effects of PEDF peptides in retinal cells continued to be investigated. The cone photoreceptor-like cell line, 661W, was used to evaluate in vitro protective effect of PEDF and PEDF derived peptides against experimentally-induced cell death. Cell death was induced by treatments with a phosphodiesterase inhibitor Zaprinast, an ionophore A23187, an oxidative stressor sodium iodate, hyperglycemia injury or by exposure to damaging light (>20,000 lux). Cell viability and death were assessed in treated cells exposed to different concentration of the effectors for different time periods. Dose response was assayed for PEDF and peptide protective activities against sodium iodate exposure. RNA from 661W exposed to sodium iodate and treated with PEDF and 44mer were isolated for Bax and Bcl2 expression determination by RT-PCR and for RNAseq analyses. Protection against cytotoxicity in vitro by chronic sodium iodate-mediated injury on RPE cells was further evaluated with PEDF. This method serves to evaluate protective factors against oxidative stress. Cytotoxicity curves of RPE cells with sodium iodate were generated. The antioxidant PEDF and the 44mer peptide from its neurotrophic region were used to optimize protection against sodium iodate-mediated cytotoxicity. Time course of protection with a given concentration of PEDF and then concentration response for protection at a given incubation time were performed. The survival signaling pathways that prevent oxidative stress by PEDF in RPE were explored, e.g., ERK1/2, mTOR, Akt, Bax, Bcl2 by RT-PCR of RNA and western blotting of protein cell extracts. The survival effects of PEDF in retinal photoreceptors during development in vitro were continued to be investigated. Pure neuronal cultures were prepared from retinas of rats at postnatal day 1, and PEDF-R protein was detected in the cells by western blotting. Polarization of rhodopsin in cells treated with PEDF peptides was evaluated. Human recombinant PEDF protein versions were purified from conditioned media of stably transfected HEK293-Ebna cells with PEDF expression vectors at milligram amounts for crystallization studies. Optimization of production and purification of large amounts of several truncated versions of the human recombinant PEDF-R protein was performed. Quality and recovery of the recombinant proteins was determined by protein analyses, such as SDS-PAGE, western blot, spectrophotometry. PEDFH105A and a synthetic peptide from PEDF-R termed P1 were set for co-crystallization by hanging drop vapor diffusion crystallography. We continued a study to elucidate the role of PEDF-R in the phagocytosis of photoreceptor outer segments (POS). Comparison of phagocytosis activity of RPE cells cultured on transwell membranes vs. plastic was performed. Distribution of PNPLA2 protein, and phagosomal and lysosomal markers was evaluated by immunofluorescence and confocal microscopy in RPE cells undergoing phagocytosis. PNPLA2 mRNA levels following POS addition to the ARPE-19 cell cultures were measured. siRNA technology was used to transiently silence PNPLA2 in the ARPE-19 cell lines and its silencing efficiency was evaluated by RT-PCR and western blot. The mRNA levels of phagocytosis-related genes were assessed in transiently transfected cells with silencing vector following POS addition. We conjugated POS with fluorescein isothiocyanate and used them to assay binding and internalization in control and transiently silenced PNPLA2 ARPE-19 cells. Pulse-chase experiments were also performed and degradation of POS by ARPE-19 cells was followed by free fatty acid production, beta-hydroxybutyrate release, and immunoreactive rhodopsin residual levels. Intracellular neutral lipid accumulation following POS addition in transiently transfected cells was assessed by BODIPY staining under the microscope. The accumulation of peroxidized lipids following POS treatment was assayed using BODIPY C11 dye. An RNA profile of fatty acid metabolism-related genes revealed that PNPLA2 silencing regulated expression of several genes, which lead to further analyses. Comparison of expression of genes of interest (ACAD9, FASN and CPT1C) was further assessed by determining protein levels by western blot. Given that PEDF-R is activated upon its interaction with PEDF, which leads to hydrolysis of phospholipids and triglycerides, the role of the PNPLA2 gene in lipophagy and autophagy in the retina and phagocytosis by RPE begun to be elucidated using tissue derived from human induced pluripotent stem cells (iPSCs). Using gene editing technology, we knocked out PNPLA2 in control iPSC lines using CRISPR-Cas9 technology. Expression of PNPLA2 was evaluated by TaqMan at the mRNA level and absence of protein was confirmed by western blotting. The association of autophagy and inflammasomes is important in aging, in which deterioration in the autophagic capacity and enhanced cellular stress results in inflammasome activation. We used induced pluripotent stem cell (iPSCs) lines from three control individuals and three individuals with age-related macular degeneration. These lines were checked for all pluripotency markers and they had a normal karyotype. Co-cultures of RPE cells and organoids can provide information of the role of autophagy in AMD and how inflammasome is regulated by autophagy. iPSCs were differentiated into RPE cells, and the levels of several specific protein markers were evaluated: Tyrosinase Related Protein 1 (TYRP1) protein levels were evaluated by western blotting, Melanocyte Inducing Transcription Factor (MITF) and premelanosome protein (PMEL17) levels were determined using immunofluorescence staining. In addition, PEDF levels were quantified in media from the apical and basal compartment of trans wells containing iPSC-derived RPE cells to determine their polarization. At the same time, the iPSCs were differentiated into retina organoids/ photoreceptors. Horse shoe eye cups were dissected, and they were kept in ultra-low adhesion plates for another 3 weeks. The presence of pigmented RPE cells and photoreceptors were observed. Co-culture between microglia and retina organoids can provide relevant tools to study the distribution of iMicroglia in the retina and activation of inflammasome in microglia in the presence of oxidative stress. The iPSCs were also differentiated into iPSCs-derived microglia. Hematopoetic stem cells were produced from iPSCs. CD34+ cells were cultured in defined microglia media for 12 days and then they were differentiated into microglia cells. Microglia markers (Purinergic Receptor P2Y12, P2RY12; Triggering receptor expressed on myeloid cells 2, TREM2; receptor for the fractalkine ligand, CXC3CR1; and Proto-oncogene tyrosine-protein kinase MER, Mertk) and their function (phagocytosis, chemotaxis and response to inflammation) were evaluated.
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