RESUMO
Adeno-associated virus (AAV) vector-mediated delivery of inhibitors of blood-retinal barrier breakdown (BRBB) offers promise for the treatment of diabetic macular edema. Here, we demonstrated a reversal of blood-retinal barrier pathology mediated by AAV type 2 (AAV2) vectors encoding vasoinhibin or soluble VEGF receptor 1 (sFlt-1) when administered intravitreally to diabetic rats. Efficacy and safety of the AAV2 vasoinhibin vector were tested by monitoring its effect on diabetes-induced changes in the retinal vascular bed and thickness, and in the electroretinogram (ERG). Also, the transduction of AAV2 vectors and expression of AAV2 receptors and co-receptors were compared between the diabetic and the non-diabetic rat retinas. AAV2 vasoinhibin or AAV2 sFlt-1 vectors were injected intravitreally before or after enhanced BRBB due to diabetes induced by streptozotocin. The BRBB was examined by the Evans blue method, the vascular bed by fluorescein angiography, expression of the AAV2 EGFP reporter vector by confocal microscopy, and the AAV2 genome, expression of transgenes, receptors, and co-receptors by quantitative PCR. AAV2 vasoinhibin and sFlt-1 vectors inhibited the diabetes-mediated increase in BRBB when injected after, but not before, diabetes was induced. The AAV2 vasoinhibin vector decreased retinal microvascular abnormalities and the diabetes-induced reduction of the B-wave of the ERG, but it had no effect in non-diabetic controls. Also, retinal thickness was not altered by diabetes or by the AAV2 vasoinhibin vector. The AAV2 genome, vasoinhibin and sFlt-1 transgenes, and EGFP levels were higher in the retinas from diabetic rats and were associated with an elevated expression of AAV2 receptors (syndecan, glypican, and perlecan) and co-receptors (fibroblast growth factor receptor 1, αvß5 integrin, and hepatocyte growth factor receptor). We conclude that retinal transduction and efficacy of AAV2 vectors are enhanced in diabetes, possibly due to their elevated cell entry. AAV2 vectors encoding vasoinhibin and sFlt-1 may be desirable gene therapeutics to target diabetic retinopathy and macular edema.
Assuntos
Proteínas de Ciclo Celular/genética , Dependovirus/genética , Diabetes Mellitus Experimental/terapia , Retinopatia Diabética/terapia , Terapia Genética , Retina/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Barreira Hematorretiniana , Vetores Genéticos , Proteoglicanas de Heparan Sulfato/análise , Masculino , Ratos , Ratos Wistar , EstreptozocinaRESUMO
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.