RESUMEN
BACKGROUND: The inducible cyclooxygenase-2 (COX-2) enzyme is upregulated in inflammatory diseases, as well as in epithelial cancers, and has an established role in angiogenesis and tissue repair. OBJECTIVE: Because of these physiological effects and the widespread use of the selective COX-2 inhibitor, celecoxib, we wanted to determine if inhibition of COX-2 would affect incisional skin wound healing. METHODS: Using a cutaneous full-thickness, sutured, incisional wound model in hairless SKH-1 mice, we evaluated the role of COX-2 in the wound healing process by comparing the effects of a nonselective COX inhibitor, diclofenac, with a selective COX-2 inhibitor, SC-791. Healing was monitored for up to 28 days postincision histologically and for recovery of wound strength. RESULTS: COX-2 expression was observed over the first week of healing, peaking at day 3 and was not affected by treatment with the selective COX-2 or nonselective COX inhibitors. Infiltrating macrophages, as well as keratinocytes and dermal fibroblasts at the wound site, expressed COX-2. Neither selective COX-2, nor nonselective COX inhibition had a significant effect on the macroscopic or microscopic morphology of the wounds, whereas dexamethasone treatment resulted in epidermal and granulation tissue atrophy. In addition, neither selective COX-2, nor nonselective COX inhibition altered keratinocyte proliferation and differentiation, dermal angiogenesis or the recovery of wound tensile strength, whereas dexamethasone reduced the tensile strength of the wounds by 30-38% throughout the healing period. CONCLUSIONS: These data indicate that selective COX-2 inhibition does not affect the healing of surgical skin wounds.
Asunto(s)
Inhibidores de la Ciclooxigenasa/metabolismo , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Piel/lesiones , Cicatrización de Heridas/fisiología , Animales , Antiinflamatorios/farmacología , Western Blotting , División Celular , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dexametasona/farmacología , Femenino , Hibridación in Situ/métodos , Queratinocitos/fisiología , Ratones , Ratones Endogámicos , Ratones Desnudos , Pruebas de Precipitina , Piel/efectos de los fármacos , Piel/enzimología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Human colon fibroblasts (HCF) produce tissue-type plasminogen activator (t-PA) in culture, but after 24-48 h, t-PA ceases to accumulate in the medium. Here, we report negative feedback regulation of t-PA expression, exerted by t-PA or complexes of t-PA with its physiological inhibitor, plasminogen activator inhibitor type 1 (PAI-1). Inhibition of t-PA expression could be induced by addition of exogenous t-PA or t-PA.PAI-1 complexes and reversed by monoclonal antibody directed against the active site of t-PA. Analysis of metabolically radiolabeled protein and cellular mRNA showed that both t-PA protein and mRNA levels declined considerably after 24 h. When 125I-labeled t-PA or t-PA.PAI-1 complexes were incubated with HCF, monensin-inhibitable endocytosis and catabolism were observed. The low density lipoprotein receptor-related protein (LRP) was found to be expressed by HCF and to mediate these events. Addition of the 39-kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, removed the block to t-PA expression and restored its accumulation in the medium. Moreover, RAP completely prevented the degradation of exogenous 125I-labeled t-PA by HCF, suggesting that LRP is the endocytic receptor for t-PA in these cells. These results demonstrate that cellular modulation of t-PA expression in HCF involves LRP receptor-mediated clearance of t-PA. This LRP receptor-mediated event results in down-regulation of t-PA expression at the mRNA level.
Asunto(s)
Colon/enzimología , Regulación Enzimológica de la Expresión Génica , Receptores Inmunológicos/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Células Cultivadas , Colon/citología , Endocitosis/efectos de los fármacos , Espacio Extracelular/enzimología , Fibroblastos/enzimología , Complejo Antigénico de Nefritis de Heymann , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Glicoproteínas de Membrana/metabolismo , Monensina/farmacología , Transcripción GenéticaRESUMEN
Oxidized low density lipoprotein (LDL) may be of central importance in triggering atherosclerosis. One potential pathway involves the production of nitric oxide (NO) by vascular wall endothelial cells and macrophages. NO reacts with superoxide to form peroxynitrite (ONOO-), a potent agent of LDL oxidation in vitro. ONOO- nitrates the aromatic ring of free tyrosine to produce 3-nitrotyrosine, a stable product. To explore the role of reactive nitrogen species such as ONOO- in the pathogenesis of vascular disease, we developed a highly sensitive and specific method involving gas chromatography and mass spectrometry to quantify 3-nitrotyrosine levels in proteins. In vitro studies demonstrated that 3-nitrotyrosine was a highly specific marker for LDL oxidized by ONOO-. LDL isolated from the plasma of healthy subjects had very low levels of 3-nitrotyrosine (9 +/- 7 micromol/mol of tyrosine). In striking contrast, LDL isolated from aortic atherosclerotic intima had 90-fold higher levels (840 +/- 140 micromol/mol of tyrosine). These observations strongly support the hypothesis that reactive nitrogen species such as ONOO- form in the human artery wall and provide direct evidence for a specific reaction pathway that promotes LDL oxidation in vivo. The detection of 3-nitrotyrosine in LDL isolated from vascular lesions raises the possibility that NO, by virtue of its ability to form reactive nitrogen intermediates, may promote atherogenesis, counteracting the well-established anti-atherogenic effects of NO.
Asunto(s)
Arterias/metabolismo , Arteriosclerosis/metabolismo , Lipoproteínas LDL/metabolismo , Nitrógeno/metabolismo , Arterias/patología , Radicales Libres , Humanos , Técnicas In Vitro , Oxidación-Reducción , Albúmina Sérica Bovina/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
In this study, we evaluated the ability of low molecular weight manganese-based superoxide dismutase mimetics to attenuate neutrophil-mediated oxygen radical damage to human aortic endothelial cells in vitro. Human neutrophils, when exposed to tumor necrosis factor-alpha and the complement compound C5a, induced endothelial damage assessed by the release of 51Cr into the medium. This damage correlated with the amount of superoxide generated by neutrophils. Three superoxide dismutase mimetics, with catalytic rate constants for superoxide dismutation ranging from 4 to 9 x 10(7) M-1 S-1, inhibited neutrophil- or xanthine oxidase-mediated endothelial cell injury in a concentration-dependent manner. A similar manganese-based compound with no detectable superoxide dismutase activity was ineffective in inhibiting injury. Fluorescent studies of the neutrophil respiratory burst showed that the superoxide dismutase mimetics were protective without interfering with the generation of superoxide by activated neutrophils. Catalase, elastase inhibitors, and desferrioxamine mesylate (an iron chelator and hydroxyl radical scavenger) were not protective against cell injury. This investigation demonstrates that neutrophil-mediated human aortic endothelial cell injury in vitro is mediated by the superoxide anion and that low molecular weight manganese-based superoxide dismutase mimetics are effective in abrogating this damage.
Asunto(s)
Antioxidantes/farmacología , Complemento C5a/toxicidad , Endotelio Vascular/patología , Etilenodiaminas/farmacología , Manganeso/farmacología , Neutrófilos/fisiología , Compuestos Organometálicos/farmacología , Superóxido Dismutasa/farmacología , Factor de Necrosis Tumoral alfa/toxicidad , Aorta , Catalasa/farmacología , Células Cultivadas , Deferoxamina/farmacología , Endotelio Vascular/citología , Humanos , Cinética , Neutrófilos/efectos de los fármacos , Oligopéptidos/farmacología , Elastasa Pancreática/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacología , Xantina Oxidasa/toxicidadRESUMEN
The contribution of xanthine oxidoreductase (XDH + XO) to the extracellular release of hydrogen peroxide (H2O2) and intracellular H2O2 concentration in cultured bovine aortic endothelial cells (BAEC) was determined. Intracellular H2O2 concentration was measured by the aminotriazole-mediated inactivation of catalase, while extracellular H2O2 release was measured by the horse-radish peroxidase-mediated oxidation of p-hydroxyphenyl acetic acid to a fluorescent dimer. Supplementation of reaction systems with xanthine did not increase H2O2 production by cells. Inhibition of XO activity with allopurinol did not decrease either intracellular concentrations or the extracellular release of H2O2. Similarly, inactivation of XO by culture of cells with tungsten did not have any effect on intracellular levels of H2O2, while it increased extracellular release of H2O2 by 86 and 103% from cells cultured in Medium 199 (M199) and Dulbecco's modified Eagle's medium (DMEM), respectively. Cells cultured in DMEM had an average of 8 times greater XDH + XO specific activity, compared to M199 cultured cells, and had a threefold greater rate of release of H2O2 than M199-grown cells. However, DMEM-cultured cells did not have a greater rate of myxothiazole-resistant respiration, suggesting that this increase in H2O2 release comes from sources other than XO. These results show that cellular XO does not contribute significantly to basal H2O2 production in bovine endothelial cells. Analysis of XDH + XO activity of endothelial cells derived from vessels of various species showed a relatively low specific activity of this potential oxidant source in human-derived cells compared with cells cultured from other species such as rodents.
Asunto(s)
Endotelio Vascular/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Xantina Oxidasa/metabolismo , Animales , Aorta/metabolismo , Bovinos , Células Cultivadas , Glucosa/metabolismo , Glucosa Oxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Cinética , Consumo de Oxígeno , Superóxidos/metabolismo , Xantina Deshidrogenasa/metabolismoRESUMEN
By stopped-flow kinetic analysis, we have directly evaluated the superoxide dismutase (SOD) activity of a number of organic nitroxides and iron- and manganese-based complexes that have been attributed with having SOD activity based upon competition experiments with cytochrome c. In 60 mM HEPES buffer, pH 8.1, or 50 mM potassium phosphate buffer, pH 7.8, Mn(II) and manganese complexes of desferal had no detectable SOD activity by stopped-flow analysis (catalytic rate constant (kcat) < 10(5.5) M-1 s-1), whereas Mn(II) and manganese complexes of desferal inhibited the reduction of cytochrome c by superoxide generated by the xanthine/xanthine oxidase system. Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (FeTPEN) was eight times more active than Fe(III)-tris[N-(2-pyridylmethyl)-2-aminoethyl]amine(Fe-TPAA) in the cytochrome c assay, but only FeTPAA catalyzed the first-order decay of superoxide (kcat = 2.15 x 10(6) M-1 s-1) by stopped-flow. Fe(III)-tetrakis(4-N-methylpyridyl)porphine (FeTMPP) was active at low micromolar concentrations in both the cytochrome c and stopped-flow assays. At high micromolar concentrations, the organic nitroxides 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPOL) were inhibitory in the cytochrome c assay, but showed no detectable SOD activity by stopped-flow. None of the tested compounds inhibited xanthine oxidase activity as shown by the measurement of urate production. Under the conditions of the cytochrome c assay, FeTPEN, TEMPO, and TEMPOL oxidized reduced cytochrome c which rationalizes the false positives for these compounds in this assay. The inhibitory activities of Mn(II) and the manganese desferal complexes in the cytochrome c assay appear to be due to a stoichiometric, not catalytic, reaction with superoxide as catalytic amounts of these agents do not induce a first-order decay of superoxide as shown by stopped-flow.
Asunto(s)
Óxidos N-Cíclicos/química , Compuestos Férricos/química , Compuestos Ferrosos/química , Manganeso/química , Superóxido Dismutasa/química , Superóxidos/química , Grupo Citocromo c/química , Técnicas In Vitro , Cinética , Oxidación-Reducción , Marcadores de SpinRESUMEN
The drastically reduced virus yields obtained from monkey cells abortively infected with adenovirus 2 (Ad2) have been attributed primarily to a severe decrease in the accumulation of the virion protein fiber (IV), a product of the most pomoter distal late gene family, L5. Here we report that the accumulation of virion protein IIIa, a product of the proximal late gene family, L1, is also severely depressed. In contrast, the i-leader protein LO-13.6K and L1 protein(s) 52K/55K are expressed with the same time course and in equal amounts in monkey cells abortively infected by Ad2 or productively infected by the Ad2-simian virus 40 (SV40) hybrid Ad2+ND1 or by the host range mutant Ad2+ND3 hr602. L1-52K/55K is phosphorylated in abortively infected CV-1 or CV-C monkey cells as well as in productively infected human and monkey cells. As with fiber expression, the failure to produce IIIa appears to be due partly to reduced or delayed IIIa mRNA accumulation. The small amount of IIIa protein that is synthesized in monkey cells is stable. Since the accumulation of both IIIa and fiber protein is deficient, the mechanism of abortive infection cannot be attributed solely to the absence of the auxiliary fiber leader sequences (1).
Asunto(s)
Adenovirus Humanos/genética , Proteínas de la Cápside , Familia de Multigenes , Proteínas Virales/biosíntesis , Adenovirus Humanos/crecimiento & desarrollo , Animales , Western Blotting , Línea Celular , Humanos , Peso Molecular , Fosforilación , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Proteínas Virales/genéticaRESUMEN
The adenovirus major late promoter functions at early and intermediate times to produce a limited set of mRNAs that appear in the cytoplasm of productively infected HeLa cells. These mRNAs may be translated in cell-free systems to produce two unrelated polypeptides of approximately 13,500 Mr (L0-13.5K and L0-13.6K) and a pair of related polypeptides of approximately 55,000 Mr (the L1-52K/55K proteins). Radiochemical protein sequence analysis of in vitro synthesized proteins has identified the N-terminal sequences of the L0-13.5K and L0-13.6K proteins (J. B. Lewis and C. W. Anderson (1983), Virology 127, 112-123). Additional sequence analyses confirmed the identification of the open reading frame for the L0-13.5K protein, and identified the ATG encoded by nucleotides 11,040 to 11,042 from the left end of the adenovirus genome as the initial codon of the L1-52K/55K protein. Antisera raised against synthetic peptides homologous to these three amino termini were used to demonstrate the presence of the L0-13.5K protein, the L0-13.6K protein, and the L1-52K/55K proteins in extracts of HeLa cells infected by adenovirus 2. The L0-13.5K protein was detected at early, intermediate, and late times after infection. The L0-13.6K and L1-52K/55K proteins were detected only at late times. Immunofluorescence microscopy indicated that the L0-13.6K protein is distributed around the periphery of the nucleus and along fibers running the length of the cell. Nonpermeabilized infected cells were stained by anti-L0-13.6K peptide serum at a single spot on the cell surface. Neither the L0-13.6K nor the L1-52K/55K proteins were detected in purified virus.
Asunto(s)
Adenovirus Humanos/genética , Genes Virales , Genes , Regiones Promotoras Genéticas , ARN Mensajero/genética , Transcripción Genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Anticuerpos , Técnica del Anticuerpo Fluorescente , Células HeLa/citología , Humanos , Inmunoelectroforesis , Fragmentos de Péptidos/análisis , Tripsina , Proteínas Virales/aislamiento & purificación , Virión/genéticaRESUMEN
Four independent, spontaneous mutants of the adenovirus type 2-simian virus 40 hybrid Ad2+ND3 that allow efficient growth in monkey cells were isolated previously (C. W. Anderson, Virology 111:263-269, 1981). All four mutations have been mapped within the coding sequence for the adenovirus DNA-binding protein by marker rescue analysis. DNA sequence analysis of a region of ca. 1,000 base pairs shown by marker rescue to contain the host range mutations demonstrated that the host range mutant hr602 differs from its parent, Ad2+ND3, at only a single nucleotide. Mutant hr602 has a thymine in place of a cytosine at the first position of the 130th codon, as measured from the initiation site for the DNA-binding protein. This change results in the replacement of a histidine by a tyrosine in mutant hr602 DNA-binding protein. Each of the other three Ad2+ND3 host range mutants have exactly the same nucleotide alteration as does hr602. This same nucleotide change was recently reported for a similarly derived host range mutant of adenovirus 5.
Asunto(s)
Adenovirus Humanos/genética , ADN Helicasas/genética , Genes Virales , Virus 40 de los Simios/genética , Proteínas Virales/genética , Adenovirus Humanos/fisiología , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , ADN Viral , Proteínas de Unión al ADN , Marcadores Genéticos , Mutación , Recombinación Genética , Virus 40 de los Simios/fisiologíaRESUMEN
The reaction of foot-and-mouth disease virus (FMDV) with 12S subunit/140S virion cross-reactive (sensitizing) antibody was studied in order to elucidate the requirements for neutralization versus sensitization. The presence of sensitizing antibody in immune serum caused an atypical in vitro neutralization response curve and a non-neutralized fraction. Cell-associated (cytophilic) antibody was not present in the system. Dissociation of the immune complex was not a factor and sensitized virus adsorbed to host cells via the regular virus receptor site(s). This finding led to the conclusion that sensitizing antibody is specific for non-critical sites. Dosing of the neutralization reaction mixtures with fractionated antibody of alternative antigenic specificities had an antagonistic effect on the neutralization response, suggesting steric hindrance. Cell receptor sites were a factor in sensitization since different host systems had different susceptibilities for sensitized antigen. The results suggest that in vitro neutralization of FMDV requires the attachment of multiple antibody molecules as proposed by the multi-hit theory of neutralization. The in vitro measurement of serum neutralizing activity as an indication of the in vivo immune response is discussed.
Asunto(s)
Aphthovirus/inmunología , Anticuerpos Antivirales/inmunología , Complejo Antígeno-Anticuerpo , Relación Dosis-Respuesta Inmunológica , Receptores Virales , Replicación ViralRESUMEN
The in vitro interaction of foot-and-mouth disease virus (FMDV) with an immune serum resulted in a fraction of virus which failed to be neutralized. This inability of antibody to neutralize the entire population of a virus preparation was studied with emphasis on the antigenic specificity of the antibody-virus reaction. Antibody to FMDV recognized multiple antigenic determinants. Immunoabsorbent fractionation of the serum into 12S subunit cross-reactive and 140S virion-specific antibody revealed that these multiple antigenic determinants are factors in determining the neutralizing ability of the antibody. Antibody specific to the infective 140S virion neutralized virus effectively, whereas antibody reactive with both the 140S virion and 12S non-infective component did so ineffectively. Neutralization was independent of viral aggregation, strain, or type heterogeneity, dissociation of the immune complex, heterogeneity of antibody class, or incubation time. The non-neutralized fraction of virus was not due to insufficient antibody in the system, was demonstrated to be complexed with antibody ('sensitized') and could be neutralized with anti-globulin serum. The findings demonstrate the heterogeneity of antibody specificity of FMDV in serum preparations and relate the importance of antibody specificity to the neutralization of virus in vitro.