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Azucena H Kakazu, Jorgelina Muriel Calandria, Jiucheng He, Haydee E P Bazan; Pigment epithelial derived factor (PEDF) and 44-mer and 34-mer PEDF peptides in the presence of docosahexaenoic acid (DHA) selectively increase neurotrophins and related genes in mouse epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4909.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies in our laboratory have shown that PEDF and the 44-mer PEDF peptide (with neurotrophic activity), but not the 34-mer PEDF (with antiangiogenic activity), in association with DHA stimulates nerve regeneration. The mechanism of axonal regeneration is not completely understood but could involve the action of neurotrophins stimulated in the target tissue. Here we investigate the pattern of neurotrophin genes activated by PEDF and its peptides (44 mer and 34 mer) plus DHA in mouse corneal epithelium.<br />
C57BL/6 mouse eyes were incubated in DMEM/F12 alone or in the presence of 50ng/ml PEDF + 50nM DHA, 5ng 34-mer PEDF+DHA, or 5ng/ml 44-mer PEDF+DHA at 370C for 3h. Epithelia were collected and RNA was purified and reverse-transcripted using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad). cDNA was amplified using SsoAdvanced SYBR Green Supermix (Bio-Rad); 10 ul of PCR mix was added to each well from the PrimePCR Custom Plate 384 wells for 62 genes (Bio-Rad). Data was collected and analyzed using CFX Manager 3.0 software. Values more than 2 fold increase were considered significant.<br />
Nine out of 62 genes were found to increase after the different treatments. Treatment with PEDF+DHA and with the two PEDF peptides in the presence of DHA increased the expression of Fgf2 gene between 7 and 12 fold with respect to control. This gene encodes the synthesis of fibroblast growth factor-2 (FGF-2). The expression of the Ntf3 gene was increased 6 times when the corneas were incubated in PEDF+DHA, but was not stimulated in the presence of the PEDF derivatives. Expression of Npff and Ppy1 genes increased more than 2 fold in the presence of PEDF+DHA or the 44-mer PEDF+DHA.<br />
Several genes that have neurotrophic activities and have not been described previously were found in the corneal epithelium when incubated with PEDF, the 44-mer PEDF or the 34-mer PEDF in the presence of DHA. Fgf2 expression increased in the three conditions. In the cornea, FGF-2 accelerated epithelial wound healing. Our previous studies show that PEDF+DHA treatment increases proliferation of epithelial cells and wound healing, and this function could be related to the entire PEDF molecule as well as the 34 mer peptide without neurotrophic properties.<br />
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