RESUMEN
Venous neointimal hyperplasia (VNH) causes hemodialysis vascular access failure. Here we tested whether VNH formation occurs in part due to local vessel hypoxia caused by surgical trauma to the vasa vasorum of the outflow vein at the time of arteriovenous fistula placement. Selective targeting of the adventitia of the outflow vein at the time of fistula creation was performed using a lentivirus-delivered small-hairpin RNA that inhibits VEGF-A expression. This resulted in significant increase in mean lumen vessel area, decreased media/adventitia area, and decreased constrictive remodeling with a significant increase in apoptosis (increase in caspase 3 activity and TUNEL staining) accompanied with decreased cellular proliferation and hypoxia-inducible factor-1α at the outflow vein. There was significant decrease in cells staining positive for α-smooth muscle actin (a myofibroblast marker) and VEGFR-1 expression with a decrease in MMP-2 and MMP-9. These results were confirmed in animals that were treated with humanized monoclonal antibody to VEGF-A with similar results. Since hypoxia can cause fibroblast to differentiate into myofibroblasts, we silenced VEGF-A gene expression in fibroblasts and subjected them to hypoxia. This decreased myofibroblast production, cellular proliferation, cell invasion, MMP-2 activity, and increased caspase 3. Thus, VEGF-A reduction at the time of arteriovenous fistula placement results in increased positive vascular remodeling.
Asunto(s)
Adventicia/cirugía , Derivación Arteriovenosa Quirúrgica/efectos adversos , Terapia Genética/métodos , Vectores Genéticos , Oclusión de Injerto Vascular/prevención & control , Venas Yugulares/cirugía , Lentivirus/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adventicia/metabolismo , Adventicia/patología , Animales , Apoptosis , Arterias Carótidas/cirugía , Caspasa 3/metabolismo , Movimiento Celular , Proliferación Celular , Células Cultivadas , Constricción Patológica , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Oclusión de Injerto Vascular/genética , Oclusión de Injerto Vascular/metabolismo , Oclusión de Injerto Vascular/patología , Hiperplasia , Venas Yugulares/metabolismo , Venas Yugulares/patología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Neointima , Nefrectomía , ARN Interferente Pequeño/genética , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
PURPOSE: Hypoxic conditions cause fibroblasts to differentiate into alpha smooth-muscle cell actin (α -SMA)-positive cells, i.e. myofibroblasts. This process is a hallmark of venous neointimal hyperplasia (VNH) associated with hemodialysis vascular access. The purpose of this study was to determine if blood outgrowth endothelial cells (BOEC) may reduce the conversion of fibroblasts into myofibroblasts under hypoxic conditions, and to determine the potential mechanisms involved. METHODS: An experimental model was used, in which fibroblasts and BOEC were subjected to hypoxia under contact and transwell conditions to determine if BOEC reduce the conversion of fibroblasts into myofibroblasts under hypoxic conditions. Gene expression under different conditions was performed. In addition, functional assays including cell proliferation and migration were determined. RESULTS: This study demonstrates that contact needs to occur between BOEC and fibroblasts for the reduction of the hypoxia-driven conversion of fibroblasts into α-SMA. This is associated with a decrease in several proangiogenic genes including vascular endothelial growth factor A, platelet-derived growth factor, fibroblast growth factor and matrix metalloproteinase 2 in fibroblasts in contact with BOEC when compared to fibroblasts alone. In addition, migration is significantly reduced while proliferation remains unchanged. CONCLUSION: This study helps provide rationale for using BOEC delivered to the adventitia of the outflow vein of hemodialysis vascular access to reduce VNH.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Comunicación Celular , Transdiferenciación Celular , Citocinas/metabolismo , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Actinas/metabolismo , Proteínas Angiogénicas/genética , Animales , Hipoxia de la Célula , Movimiento Celular , Proliferación Celular , Tamaño de la Célula , Células Cultivadas , Técnicas de Cocultivo , Citocinas/genética , Regulación hacia Abajo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Miofibroblastos/metabolismo , Fenotipo , Transducción de Señal , Porcinos , Factores de TiempoRESUMEN
Hemodialysis vascular access can develop venous neointimal hyperplasia (VNH) causing stenosis. Recent clinical and experimental data has demonstrated that there is increased expression of a disintegrin and metalloproteinase thrombospondin motifs-1 (ADAMTS-1) at site of VNH. The experiments outlined in the present paper were designed to test the hypothesis that targeting of the adventitia of the outflow vein of murine arteriovenous fistula (AVF) using a small hairpin RNA that inhibits ADAMTS-1 expression (LV-shRNA-ADAMTS-1) at the time of fistula creation will decrease VNH. At early time points, ADAMTS-1 expression was significantly decreased associated with a reduction in vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-9 (MMP-9) (LV-shRNA-ADAMTS-1 transduced vessels vs. controls). These changes in gene and protein expression resulted in favorable vascular remodeling with a significant increase in mean lumen vessel area, decrease in media/adventitia area, with a significant increase in TUNEL staining accompanied with a decrease in cellular proliferation accompanied with a reduction in CD68 staining. Collectively, these results demonstrate that ADAMTS-1 transduced vessels of the outflow vein of AVF have positive vascular remodeling.