RESUMEN
Increased vascular permeability associated with retinal vascular leakage is known to occur in patients with diabetes, and contributes to endothelial barrier dysfunction. The purpose of this study was to examine the effect of pigment epithelium-derived factor (PEDF) on signaling cascade in porcine retinal endothelial cells (PREC) related to permeability and angiogenesis induced by vascular endothelial growth factor (VEGF)-and interleukin-1beta (IL-1beta). PREC were exposed to VEGF, IL-1beta and PEDF at different concentrations, and in vitro permeability was assessed by solute flux assay using 70-kDa RITC-dextran. Angiogenic assays such as proliferation, migration and tube formation were determined by MTT, wound-scratch method and on-gel assay system respectively. To explore the signaling pathways behind VEGF-and IL-1beta-induced PREC permeability, an inhibitor assay was carried out using PP2, a Src kinase inhibitor. Further, Src activity was assessed by transient transfection assay using constitutively active (CA) and dominant negative (DN) Src mutants. We report that VEGF-and IL-1beta-stimulates permeability, in a dose and time-dependent manner and PEDF inhibits the VEGF-and IL-1beta-induced PREC permeability. In addition, PEDF inhibits the VEGF-and IL-1beta-induced endothelial cell proliferation, migration and tube formation. In addition, overexpression of DN Src blocked both VEGF-and IL-1beta-stimulation of permeability, proliferation and migration, while overexpression of CA Src overpowers the inhibitory action of PEDF on permeability, proliferation and migration. These results demonstrate that PEDF may inhibit the VEGF-and IL-1beta-induced permeability and angiogenesis via Src-dependent pathway.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Proteínas del Ojo/farmacología , Interleucina-1beta/farmacología , Factores de Crecimiento Nervioso/farmacología , Retina/efectos de los fármacos , Serpinas/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Permeabilidad Capilar/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Proteínas del Ojo/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Factores de Crecimiento Nervioso/metabolismo , Retina/citología , Retina/metabolismo , Serpinas/metabolismo , Porcinos , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Pigment epithelium-derived factor (PEDF) is a well-known protease inhibitor for angiogenesis in the eye, suggesting that loss of PEDF in eye is implicated in the pathogenesis of proliferative diabetic retinopathy. Since the role of PEDF in diabetic retinopathy is unclear, the effect of PEDF on different types of cells constituting the blood vessel has to be checked. Here, we have investigated the effects of PEDF under hyperglycemic conditions in retinal pericytes, isolated from goat's eye and used to analyze the signaling pathway involved. High glucose increased the apoptotic cell death and intracellular reactive oxygen species generation, which was blocked on the addition of PEDF. PEDF was found to inhibit the apoptotic cell death and protect the cells via activating the PI3K/Akt pathway, which was analyzed with dominant negative Akt and constitutively active Akt-transfected cells. These results demonstrate that PEDF protects pericytes against the high glucose-induced apoptosis and dysfunction.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas del Ojo/fisiología , Glucosa/farmacología , Factores de Crecimiento Nervioso/fisiología , Proteína Oncogénica v-akt/fisiología , Pericitos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/fisiología , Vasos Retinianos/citología , Serpinas/fisiología , Animales , Caspasa 3/metabolismo , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Genes Dominantes , Cabras , Proteína Oncogénica v-akt/genética , Pericitos/citología , Pericitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Retinal angiogenesis in diabetes may lead to visual impairment and even irreversible blindness in people of working age group worldwide. The main pathological feature of proliferative diabetic retinopathy (PDR) is hypoxia, and overproduction of growth factors like vascular endothelial growth factor (VEGF) and erythropoietin (Epo). This results in pathological proliferation of retinal endothelial cells (RECs), leading to new vessel formation (angiogenesis). Inhibition of angiogenesis is a promising strategy for treatment of PDR and other retinal neovascular disorders. Pigment epithelium-derived factor (PEDF), a 50-kDa protein secreted by retinal pigment epithelium, inhibits the growth of new blood vessel induced in the eye in a variety of ways with a yet elusive mechanism. Here, we investigated the possible mechanism by which PEDF inhibits VEGF- and Epo-induced angiogenic effects in RECs is mediated through PI3K/Akt pathway. PEDF treatment induced the apoptosis in RECs by activating caspase-3 and DNA fragmentation. We found a dose-dependent increase in cell survival with VEGF or Epo, which was attenuated in the presence of PEDF. In addition, PEDF significantly (P < 0.05) inhibited migration and in vitro tube formation in RECs in the presence of VEGF as like PI3K/Akt inhibitor. Of interest, PEDF effectively abrogated VEGF-mediated phosphorylation of PI3K/Akt. Further studies using RECs transfected with constitutively active and dominant-negative forms of Akt suggest that PEDF could inhibit VEGF- and also Epo-induced angiogenesis by disruption of PI3K/Akt signaling.