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Preeti Subramanian, Jeanee Bullock, Paige Winokur, Veronique Arocas, S. Patricia Becerra; Protease Nexin-1 (PN-1): A Novel Survival Factor for Retina Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1738. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Protease Nexin-1 (PN-1) is a member of the serine protease inhibitor (serpin) superfamily. It has demonstrable neurotrophic effects in the brain. Its gene SERPINE2 is expressed in the retina. PN-1 inhibits serine protease thrombin. Recently it was shown that PN-1 inhibits angiogenesis in the retina. The neurotrophic capacities of PN-1 in the retina have not been evaluated. The purpose of this study was to investigate the efficacy of the PN-1 in protecting the retina.
Rat retinal progenitor R28 cells and human ARPE-19 cells were cultured. PN-1 was analyzed by western blots and probed vs anti-PN-1 antibody. Bacterially-derived human PN-1 and RS10 and PN-1-derived 17mer peptide and mammalian-derived pigment epithelium-derived factor (PEDF) were used. TUNEL assays were performed to quantitate cell-death. Binding assays to ligand binding domain of PEDF-receptor was done by peptide affinity chromatography. RT-PCR was performed for Bcl-2.
A PN-1 immuno-reactive band was detected in total lysates from ARPE-19 cells but was undetectable in media or washes of cells with high ionic strength buffers. We tested PN-1 and RS10, a modified PN-1 that lacks the ability to inhibit serine proteases, for survival of retina cells. Both versions of PN-1 (at 2.5 nM and 10 nM) decreased the percentage of TUNEL positive cells in R28 cells relative to untreated cells. PN-1 is similar to PEDF, a neurotrophic factor for the retina. PN-1-derived 17mer peptide was designed from the alignment with the neurotrophic region of PEDF. PN1-17mer also decreased the percentage of TUNEL positive cells relative to untreated cells, like full-length PN-1 and PEDF. Percentage of binding to PEDF-receptor of fluoresceinated PN-1-17mer and fluoresceinated PEDF-17 mer peptides was 11% and 45% of the total ligand input, respectively. Expression of the anti-apoptotic Bcl-2 gene increased upon treatment with PN-1 for 6 h relative to untreated and was similar to treatments with fetal bovine serum (FBS).
PN-1 is a survival factor for retina cells in culture and its mechanism of action is independent of inhibition of serine protease inhibition. A small region towards the amino terminus confers the survival activity to the PN-1 polypeptide.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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