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Puran Bora, Nalini Bora, Valeriy Lyzogubov; Adiponectin Peptide II (APNpII) effect on human ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4581.
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We showed before that adiponectin peptide II (APNpII) has anti-angiogenic effect in mouse model of choroidal neovascularization (CNV). We also observed increased expression of adiponectin receptors 1 (AdipoR1) in laser injured area and retinal pigment epithelim (RPE) in mice. The aim of this study was to investigate the effect of APNpII on human ARPE-19 cells.
Human ARPE-19 cells were grown on Transwell 24 mm diameter, 0.4 µm pore size tissue culture polyester membrane till confluence. We treated the cells with APNpII (20 μg/mL) or with medium for 8 days. Wounding was induced by scratching the cell monolayer with pipette tip. Concentration of vascular endothelial growth factor (VEGF) and chemokine (C-C motif) ligand 2 (CCL2) in supernatants from upper and lower chamber was measured using ELISA. The area of the wound was measured by using ImageJ program. We stained ARPE-19 cells for AdipoR1 and proliferating cell nuclear antigen (PCNA) using immunohistochemistry (IHC) and investigated expression of AdipoR1 and PCNA using laser confocal microscopy. Statistical analysis was carried out using analysis of variance (ANOVA).
We detected AdipoR1 not only on the surface of ARPE-19 cells but also in the cytoplasm (predominately in perinuclear zone) and inside nucleus. Treatment with APNpII did not affect expression of AdipoR1. However, mechanical damage of ARPE-19 cells resulted in increase of AdipoR1 expression in PCNA positive cells. APNpII reduced concentration of VEGF and/or CCL2 in supernatant before wounding (20% and 34%, respectively) and 48 hours after wounding (26% and 25%, respectively). APNpII accelerated healing of ARPE-19 monolayer after mechanical damage. Area of damage in APNpII treated group was 38% less in 24 hours and 59% less in 48 hours after injury compared to control. Expression of PCNA in wounded area was 20% more in APNpII treated ARPE-19 cells compared to control group.
RPE cells are potential target for anti-angiogenic therapy. APNpII may be used for inhibiting the VEGF and CCL2 secretion by RPE cells. APNpII peptide may have therapeutic use for AMD treatment.
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