RESUMEN
PURPOSE: To investigate the correlation between the presence of the inactive cathepsin D (CatD) and retinal changes in mcd2/mcd2 transgenic mice. METHODS: Computational modeling was used to examine whether CatD mutants maintain competitive substrate binding. D407 cells were transfected with pcDNACatDM1 or pcDNACatDM2, containing procathepsin D (pro-CatD) with 6-bp (CatDM1) or 12-bp (CatDM2) deletions, respectively, flanking the pro-CatD cleavage site, and the aspartic protease activity of the transfected cells was measured. Subsequently, transgenic mice (mcd2/mcd2) containing CatDM2 were generated. Relative transgene copy number and transcript levels in the previously produced mcd/mcd (carrying CatDM1) and mcd2/mcd2 mice were measured by quantitative real-time PCR. Western blot analysis and aspartic protease activity were used to characterize the mutated proteins. Retinal changes were described by using color fundus photography and fluorescein angiography, histology, immunohistochemistry, and electron microscopy. RESULTS: Computational modeling of the CatDM1 and CatDM2 structures indicated that the substrate binding site was not altered. There was limited or no aspartic protease activity associated with CatDM1 and CatDM2 proteins, respectively. Mcd2/mcd2 animals contained a higher amount of inactive CatD than mcd/mcd or wild-type mice. Retinal abnormalities in mcd2/mcd2 mice developed at 3 months of age, earlier than in mcd/mcd mice. These changes included hypopigmentation, hyperfluorescence, retinal pigment epithelial (RPE) cell depigmentation or clumping, cell proliferation, and pleomorphism. Proliferating cells were identified as being of RPE origin. CONCLUSIONS: This study demonstrated a correlation between the presence of the inactive CatD in RPE cells and the development of ophthalmoscopic, cellular, and histologic changes in the retina.
Asunto(s)
Catepsina D/fisiología , Degeneración Macular/enzimología , Epitelio Pigmentado Ocular/enzimología , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Western Blotting , Catepsina D/química , Simulación por Computador , Precursores Enzimáticos/fisiología , Angiografía con Fluoresceína , Eliminación de Gen , Humanos , Inmunohistoquímica , Degeneración Macular/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estructura Molecular , Epitelio Pigmentado Ocular/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Successful clinical transplantation of whole skeletal muscles can be limited by impaired muscle revascularization and regeneration. The aim of this study was to enhance the revascularization (and hence speed of regeneration) of transplanted whole muscles by transducing muscles with the vascular endothelial growth factor (VEGF) gene before transplantation, using a recombinant adeno-associated virus (rAAV). The rAAV encoding VEGF and green fluorescent protein (GFP) (rAAV.VEGF.GFP) was injected into the tibialis anterior muscles of adult BALB/c mice. One month after injection whole muscle autotransplantation was performed. Muscles were sampled 7 days after autografting. GFP expression was examined as an indicator of persistent transgene expression after grafting, and immunohistochemistry was used to identify VEGF, blood vessels, and newly formed myotubes. After grafting, GFP expression persisted only in a few surviving myofibers in the periphery of rAAV.VEGF.GFP-pretreated muscles, although abundant VEGF expression was seen in myogenic cells in all grafted muscles. Quantitative analysis demonstrated that, although only small numbers of rAAV.VEGF.GFP-transduced myofibers were present, whole muscle grafts preinjected with rAAV.VEGF.GFP were significantly more vascular than saline-injected and uninjected control muscle grafts. Furthermore, rAAV.VEGF.GFP-injected whole muscle transplants were further advanced in terms of regeneration (myotube formation) compared with the uninjected control muscle transplants. This study clearly shows that rAAV-mediated VEGF expression persists only in myofibers that survive the necrosis induced by muscle transplantation; however, this amount of VEGF results in significantly increased revascularization and regeneration of whole muscle transplants.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Terapia Genética , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/genética , Músculo Esquelético/fisiología , Neovascularización Fisiológica/fisiología , Regeneración/fisiología , Animales , Dependovirus , Factores de Crecimiento Endotelial/metabolismo , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Linfocinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/trasplante , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Developed over the past two decades, the antisense strategy has become a technology of recognised therapeutic potential, and many of the problems raised earlier in its application have been solved to varying extents. However, the adequate delivery of antisense oligodeoxynucleotides to individual cells remains an important and inordinately difficult challenge. Synthetic polymers appeared on this scene in the middle 1980s, and there is a surprisingly large variety used or proposed so far as agents for delivery of oligodeoxynucleotides. After discussing the principles of antisense strategy, certain aspects of the ingestion of macromolecules by cells, and the present situation of delivery procedures, this article analyses in detail the attempts to use synthetic polymers as carrier matrices and or cell membrane permeabilisation agents for delivery of antisense oligodeoxynucleotides. Structural aspects of various polymers, as well as the results, promises and limitations of their use are critically evaluated.