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Andrea Carvalho, Josep Badal, Miguel Zapata, Anna Salas Torras, Laura Distefano, Jose Garcia-Arumi, ; EVALUATION OF ANTIANGIOGENIC EFFECT OF SINTETIC SMALL FRAGMENTS OF PEDF. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4618.
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© ARVO (1962-2015); The Authors (2016-present)
PEDF is a glycoprotein that belongs to the serine protease inhibitors. A functional region for the inhibition of vessel sprouting and CNV resides within the 34-mer region of PEDF. Here, we examined the antiangiogenic properties of 7 small fractions of synthetic 34-mer PEDF fragments as a first step to develop an antiangiogenic treatment able to penetrate cornea and reach the retina in therapeutic levels.
Seven different fractions of synthetic PEDF were evaluated in HUVEC cultures. To evaluate the effect of the different fragments in cell migration, a wound was created in the center of a confluent culture, cells were then treated with VEGF and VEGF+PEDF fractions, healing area was quatified after 12 hours of treatment. To verify the capacity to inhibit tube formation HUVEC was co-cultured with fibroblasts and the cell medium was supplemented with VEGF+FGF-2 and VEGF+FGF-2+PEDF fractions. Cells were immunostained with VWF and images were obtained from fluorescent microscopy. The number, length and junctions of tubes, as well as the number of aggregates were measured and counted. Cell viability was measured by reduction of Alamar Blue and trypan blue staining after 12 hours of treatment. All experiments were made in duplicate and repeated 3 times. It was evaluated different concentrations of each PEDF fractions (1nM, 5nM, 10nM and 50nM) in all assays.
From the 7 fractions tested only fractions 2 (F2) and 3 (F3) were effective inhibiting cell migration p< 0,04 and 0,02 (10nM), and 0,02 (50nM) for both fractions. Tube length and number of aggregates was not significative for all fractions and concentrations tested. There were fewer tubes after treatment with F2 (p< 0,027) at 5nM, F2 (p< 0,0005) and F3 (p< 0,005) at 10nM and p< 0,05 for F2, F3, F6 and F7 at 50nM concentration. The number of tube junctions was reduced (p< 0,05) in cultures treated with all fractions and concentrations tested. Alamar Blue reduction show no difference after treatments. The was an increase in dead cells visualized by trypan blue in cells treated with F2 (p< 0,04) and 3 (p< 0,03) at 5nM, fractions 2, 3, 4 and 6 (p< 0,05) at 10nM and all fractions at 50nM
The F2 is the most effective at less concentration. It proves to inhibit tube formation and junction, endothelial migration and decrease cell viability. This result is promising for the development of an effective topical antiangiogenic, non-invasive therapy.
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