RESUMEN
ABSTRACT Copper plays a key role in angiogenesis and in the synthesis and stabilization of extracellular matrix skin proteins, which are critical processes of skin formation. We hypothesized that introducing copper into wound dressings would enhance wound repair. Application of wound dressings containing copper oxide to wounds inflicted in genetically engineered diabetic mice (C57BL/KsOlaHsd-Lepr(db)) resulted in increased gene and in situ up-regulation of proangiogenic factors (e.g., placental growth factor, hypoxia-inducible factor-1 alpha, and vascular endothelial growth factor), increased blood vessel formation (p<0.05), and enhanced wound closure (p<0.01) as compared with control dressings (without copper) or commercial wound dressings containing silver. This study proves the capacity of copper oxide-containing wound dressings to enhance wound healing and sheds light onto the molecular mechanisms by which copper oxide-impregnated dressings stimulate wound healing.
Asunto(s)
Vendajes , Cobre/farmacología , Piel/patología , Oligoelementos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Diabetes Mellitus Experimental , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Factor de Crecimiento Placentario , Proteínas Gestacionales/metabolismo , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We have recently reported an alternative cell therapy approach to induce angiogenesis. The approach is based on small organ fragments--micro-organs (MOs)--whose geometry allows preservation of the natural epithelial/mesenchymal interactions and ensures appropriate diffusion of nutrients and gases to all cells. We have shown that lung-derived MOs, when implanted into hosts, transcribe a wide spectrum array of angiogenic factors and can induce an angiogenic response that can rescue experimentally induced ischemic regions in mice. From a clinical perspective, skin-derived MOs are particularly appealing as they could readily be obtained from a skin biopsy taken from the same target patient. In the present work we have investigated the angiogenesis-inducing capacity of rabbit and human skin-derived micro-organs in vitro and in vivo. Rabbit skin MOs were implanted into homologous adult rabbits and human skin MOs were encapsulated and implanted into xenogenic mice. Skin-derived MOs, as lung-derived MOs, were found to secrete a whole array of angiogenic factors and to induce a powerful angiogenic response when implanted back into animals. We believe the approach presented suggests a novel, efficacious and simple approach for therapeutic angiogenesis.
Asunto(s)
Neovascularización Fisiológica , Trasplante de Piel , Piel/irrigación sanguínea , Adulto , Animales , Femenino , Humanos , Ratones , Persona de Mediana Edad , Conejos , Técnicas de Cultivo de Tejidos , Trasplante HeterólogoRESUMEN
Activation of macrophages leads to the secretion of cytokines and enzymes that shape the inflammatory response and increase metabolic processes. This, in turn, results in increased production of reactive oxygen species. The role of Cu/Zn superoxide dismutase (SOD-1), an important enzyme in cellular oxygen metabolism, was examined in activated peritoneal elicited macrophages (PEM) and in several inflammatory processes in vivo. LPS and TNF-alpha induced SOD-1 in PEM. SOD-1 induction by LPS was mainly via extracellular signal-regulated kinase-1 activation. Transgenic mice overexpressing SOD-1 demonstrated a significant increase in the release of TNF-alpha and of the metalloproteinases MMP-2 and MMP-9 from PEM. Disulfiram (DSF), an inhibitor of SOD-1, strongly inhibited the release of TNF-alpha, vascular endothelial growth factor, and MMP-2 and MMP-9 from cultured activated PEM. These effects were prevented by addition of antioxidants, further indicating involvement of reactive oxygen species. In vivo, transgenic mice overexpressing SOD-1 demonstrated a 4-fold increase in serum TNF-alpha levels and 2-fold stronger delayed-type hypersensitivity reaction as compared with control nontransgenic mice. Conversely, oral administration of DSF lowered TNF-alpha serum level by 4-fold, lowered the delayed-type hypersensitivity response in a dose-dependent manner, and significantly inhibited adjuvant arthritis in Lewis rats. The data suggest an important role for SOD-1 in inflammation, establish DSF as a potential inhibitor of inflammation, and raise the possibility that regulation of SOD-1 activity may be important in the treatment of immune-dependent pathologies.
Asunto(s)
Inflamación/enzimología , Inflamación/inmunología , Superóxido Dismutasa/fisiología , Administración Oral , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/farmacología , Artritis Experimental/prevención & control , Células Cultivadas , Colagenasas/metabolismo , Cobre/farmacología , Disulfiram/administración & dosificación , Disulfiram/antagonistas & inhibidores , Disulfiram/farmacología , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Factores de Crecimiento Endotelial/metabolismo , Activación Enzimática/inmunología , Femenino , Humanos , Hipersensibilidad Tardía/enzimología , Hipersensibilidad Tardía/inmunología , Inflamación/metabolismo , Inflamación/prevención & control , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lipopolisacáridos/farmacología , Linfocinas/antagonistas & inhibidores , Linfocinas/metabolismo , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Ratas , Ratas Endogámicas Lew , Superóxido Dismutasa/biosíntesis , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba/inmunología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The adiposity hormone leptin regulates food intake, body weight, reproduction and other metabolic and endocrine functions mainly through signaling to the hypothalamus. Leptin signaling to peripheral tissues other than the hypothalamus has been suggested for a number of processes such as immunity, bone metabolism, hematopoiesis, angiogenesis, and wound healing. It was previously shown that exogenously applied leptin accelerated wound healing and that leptin mRNA is expressed at the wound site, but there is no published evidence showing that it is translated into leptin protein that is available at the site of repair. To address this question we analyzed pig wound fluids collected from partial-thickness excisional wounds during the first 9 days after injury. Leptin was measured using a modified culture of HEK-293 cells, expressing both the human leptin receptor gene and the firefly luciferase gene driven by a STAT-inducible promoter. Relatively high levels of leptin activity (50-250 ng/ml) were detected in wound fluids using the leptin receptor expressing HEK-293 cells. Our results suggest that leptin is normally induced (4.8- to 10.2-fold) in wound tissue during the first few days following injury and may operate in a paracrine or autocrine circuit during the wound repair process.
Asunto(s)
Líquidos Corporales/metabolismo , Leptina/metabolismo , Piel/metabolismo , Cicatrización de Heridas , Animales , Células Cultivadas , Humanos , Riñón/citología , Luciferasas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Leptina , Piel/lesiones , PorcinosRESUMEN
Endothelial cells produce oxygen radicals spontaneously and this process is augmented by hypoxia/reoxygenation. Cu/Zn superoxide dismutase (SOD-1) is an important enzyme in cellular oxygen metabolism. To determine whether alterations in SOD-1 activity affect angiogenesis we used transgenic SOD-1 (Tg-SOD) mice with elevated level of SOD-1. Angiogenesis induced subcutaneously by bFGF in Tg-SOD mice was 3-fold higher than in control non-transgenic (ntg) mice. Oral administration of disulfiram (DSF), an inhibitor of SOD-1, inhibited angiogenesis in Tg-SOD mice as well as in CD1 nude mice. Effects of DSF on cultured cells were also tested. Application of DSF to cultured bovine capillary endothelial (BCE) cells caused inhibition of DNA synthesis and induction of apoptosis. These effects were prevented by addition of antioxidants, further indicating involvement of reactive oxygen species. DSF also reduced the level of glutathione and the production of H(2)O(2) in BCE cells. Moreover, PC12-SOD cells with elevated SOD-1 were less sensitive to DSF treatment then control cells. These data indicate that the effects of DSF are mediated by inhibition of SOD-1 activity. Tumor development is known to largely depend on angiogenesis. We found that oral administration of DSF to mice caused significant inhibition of C6 glioma tumor development and marked reduction (by 10-19-fold) in metastatic growth of Lewis lung carcinoma. The data suggest a role for SOD-1 in angiogenesis, establish DSF as a potential inhibitor of angiogenesis and raise the possibility that attenuating SOD-1 activity may be important in treatment of angiogenesis-dependent pathologies.
Asunto(s)
Neovascularización Patológica/enzimología , Superóxido Dismutasa/metabolismo , Animales , Apoptosis , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/prevención & control , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/prevención & control , Disulfiram/farmacología , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glioma/irrigación sanguínea , Glioma/prevención & control , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Etiquetado Corte-Fin in Situ , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Ratas , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/genética , Células Tumorales CultivadasRESUMEN
To add an additional aspect to the process of recognition and removal of senescent human erythrocytes from the circulation, the binding of wheat germ agglutinin (WGA) to separated young, old and sialidase-treated human erythrocytes is evaluated with the immune-electron microscopical method. WGA/gold conjugate binding to old erythrocytes was lower (27%) than to young erythrocytes and even lower following treatment with sialidase (82%), exhibiting a clustered, non-continuous labeling pattern in all three erythrocyte populations, thus showing a possible redistribution of WGA binding sites. The decrease in bound WGA/gold particles correlates well with the previously reported decrease in surface sialic acid on old erythrocytes. The binding of WGA/gold are indicative of the changes occurring on erythrocyte membrane surfaces when interacting with different agglutinins.