RESUMEN
Thermal trauma causes tissue damage by membrane destabilization and energy depletion at the cellular level, resulting in tissue necrosis and inflammation leading to delayed cell death. One therapeutic approach is to block the immediate triggering of the inflammatory cascade that results in prolonged hypermetabolic responses and immune dysfunction while promoting the expression of growth factors. In the present study, we determined hepatic gene expression responses to insulin-like growth factors-i (IGF-I) gene transfer to burned rats using high-density DNA microarray assays. The expression of 123 out of approximately 8,800 genes assayed (1.4% of total) were significantly altered. Of these, 42 genes were altered irrespective of treatment by burn trauma (p < 0.05). Changes in gene expression were confirmed by measuring mRNA levels using reverse transcription-polymerase chain reaction and protein levels by Western blot assays. DNA microarray analyses showed two major mechanisms that mediated beneficial outcomes after IGF-I gene transfer in the burned rat livers. These mechanisms were the stimulation of IGF binding protein potentiation of peripheral IGF-I and the inhibition of the burn-augmented pro-apoptotic and oxidative mitochondrial metabolites stimulated by thermal trauma.
Asunto(s)
Quemaduras/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Hígado/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Técnicas de Transferencia de Gen , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Quininógeno de Alto Peso Molecular/genética , Quininógeno de Alto Peso Molecular/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Complementario/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin.