RESUMEN
PURPOSE: Specific proteolytic cleavages of the hormone prolactin (PRL) generate vasoinhibins, a family of peptides (including 16-kDa PRL) that are able to inhibit the pathologic increase in retinal vasopermeability (RVP) associated with diabetes. Here the authors tested the ability of an adenoassociated virus type 2 (AAV2) vasoinhibin vector to inhibit vascular endothelial growth factor (VEGF)- and diabetes-induced RVP. METHODS: AAV2 vectors encoding vasoinhibin, PRL, or soluble VEGF receptor 1 (soluble FMS-like tyrosine kinase-1 [sFlt-1]) were injected intravitreally into the eyes of rats. Four weeks later, either VEGF was injected intravitreally or diabetes was induced with streptozotocin. Tracer accumulation was evaluated as an index of RVP using fluorescein angiography or the Evans blue dye method. RT-PCR verified transgene expression in the retina, and the intravitreal injection of an AAV2 vector encoding green fluorescent protein revealed transduced cells in the retinal ganglion cell layer. In addition, Western blot analysis of AAV2-transduced HEK293 cells confirmed the expression and secretion of the vector-encoded proteins. RESULTS: The AAV2-vasoinhibin vector prevented the increase in tracer accumulation that occurs 24 hours after the intravitreal injection of VEGF. Diabetes induced a significant increase in tracer accumulation compared with nondiabetic controls. This increase was blocked by the AAV2-vasoinhibin vector and reduced by the AAV2-sFlt-1 vector. The AAV2-PRL vector had no effect. CONCLUSIONS: These results show that an AAV2-vasoinhibin vector prevents pathologic RVP and suggest it could have therapeutic value in patients with diabetic retinopathy.
Asunto(s)
Permeabilidad Capilar/genética , Proteínas de Ciclo Celular/genética , Dependovirus/genética , Diabetes Mellitus Experimental/complicaciones , Retinopatía Diabética/terapia , Terapia Genética/métodos , Albúminas/farmacocinética , Animales , Permeabilidad Capilar/efectos de los fármacos , Colorantes/farmacocinética , Dextranos/farmacocinética , Diabetes Mellitus Experimental/genética , Retinopatía Diabética/genética , Modelos Animales de Enfermedad , Azul de Evans/farmacocinética , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Inyecciones Intravítreas , Masculino , Plásmidos/genética , Ratas , Ratas Wistar , Hemorragia Retiniana/inducido químicamente , Hemorragia Retiniana/genética , Hemorragia Retiniana/terapia , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Prolactin (PRL), originally associated with milk secretion, is known to have a wide variety of biological actions and diverse sites of production beyond the pituitary gland. Recent studies have demonstrated that PRL is synthesized in retinal tissue. To gain insights into the functional role of PRL in the mammalian retina, we mapped the distribution of the PRL protein and the expression and localization of the PRL receptor (PRLR) in the retina of adult rats and green monkeys. PRL was examined in retinal sections by double immunolabeling combining anti-PRL antibodies with antibodies specific for glutamine synthetase (labeling Müller cells), glial fibrillary acidic protein (labeling astrocytes), or neuronal nuclei protein (labeling neurons). PRL was detected throughout the rat retina: in the photoreceptor outer segments, Müller cells, interneurons, ganglion cells, and astrocytes. The PRLR was examined by RT-PCR, in situ hybridization, immunohistochemistry, and Western blot. The long isoform of the PRLR was localized in the photoreceptor nuclear layer, inner nuclear layer, and ganglion cell layer of rat retina. The monkey retina showed a similar distribution of PRL and PRLR immunoreactivities. These findings suggest that PRL functions as a local regulator of various cell types in the mammalian retina.
