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Anna Salas Torras, Andrea Carvalho, Ibane Abasolo, Miguel Zapata, Laura Distefano, Simo Schwartz, Jose Garcia-Arumi; In vitro studies on the antiangiogenic effects of Pigment Epithelium Derived Factor and Somatostatin. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4660.
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© ARVO (1962-2015); The Authors (2016-present)
Pigment Epithelium Derived Factor (PEDF) and Somatostatin (SST) have been demonstrated to be two of the principal natural antiangiogenic and anti-VEGF factors present in the retina. Both peptides are promising tools to be used in different strategies for a variety of retinal disorders affecting the endothelial compartment such as PDR and wet AMD. The aim of this study is to evaluate the antiangiogenic and antiproliferative properties of PEDF and SST in several in vitro models, as a preliminary proof-of-concept assay before testing their efficacy in vivo.
To test the antiangiogenic properties of PEDF and SST in vitro, two cell types were used: the ARPE-19 cell line derived from the retinal pigment epithelium, and a primary culture of human umbilical vein endothelial cells (HUVECs), as a model of vascular endothelium. Both are the main cell types affected in proliferative eye diseases. First we determined the expression of SST receptors (SSTR) in HUVECs and ARPE-19 by RT-PCR. PEDF receptors could not be verified, as they are still not identified. Antiproliferative action of PEDF and SST were tested in HUVEC and ARPE-19 cells by MTT assay after 24 and 48 h of incubation. In addition, HUVECs were used as an endothelial model to perform migration and tubule formation assays under VEGF treatment, in order to test the antiangiogenic activity of PEDF and SST.
Using RT-PCR, ARPE-19 cells were found to express SSTR-1, -2, -3 and -5, while HUVEC cells mainly express SSTR-1 and -2. In proliferation assays, SST was found to significantly inhibit ARPE-19 proliferation up to a 40% at 10-4M, (p<0,001). PEDF only inhibited proliferation of ARPE-19 cells in a 13% (10-8M, p<0,001). Both PEDF and SST showed a similar suppression of endothelial migration (35% and 39% respectively, p<0,05) in HUVEC cells treated with 25 ng/ml of VEGF. Finally, tube formation assay showed that PEDF was able to reduce the tubule length as well as the number of tubules and connections (22%, 30% and 57% respectively, p<0,05). On the other hand, SST was able to reduce the number of tubules and connections but not the tubule length (51% and 68% respectively, p<0,05).
Our data demonstrate that PEDF and SST have both antiproliferative and antiangiogenic effects in retinal pigment epithelium and endothelial cells and can be good candidates for the treatment of retino-epithelial disorders in vivo.
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