RESUMEN
We have previously demonstrated the potency of coumarinic derivatives to inhibit human leukocyte elastase. Given the anti-inflammatory activities of some coumarins, we investigated the capacity of our coumarinic derivatives to inhibit inflammation and whether their anti-elastase activity was essential for their anti-inflammatory functions. All compounds studied were coumarinic derivatives displaying differential anti-proteinase activity. Coumarinic derivatives 1, 2, and 3 efficiently inhibited human leukocyte elastase in vitro, whereas the coumarinic derivative 4 did not show inhibitory activity. The anti-inflammatory effect of these compounds and a coumarin control, scopoletin, on interleukin-6 (IL-6), tumor necrosis factor (TNF), and macrophage chemotactic protein-1 (MCP-1) release was studied using lipopolysaccharide (LPS)-stimulated alveolar macrophages. The in vivo effect of compound 2, that inhibits elastase, and compound 4, that does not show proteinase inhibition, was investigated using a mouse model of LPS-induced lung inflammation and elastase-induced acute lung injury. All investigated coumarinic derivatives, regardless of their anti-proteinase activity, significantly inhibited IL-6 and TNF production by LPS-stimulated alveolar macrophages. However, only compounds 2, 3, and 4 significantly reduced MCP-1 release. Compound 2 attenuated LPS-induced leukocyte recruitment in bronchoalveolar lavage, whereas no inhibition was observed with compound 4 devoid of elastase inhibitory capacity. Interestingly, MCP-1 level was reduced in bronchoalveolar lavage of compound 4 treated mice, whereas TNF and IL-6 levels were not modulated by coumarins. Furthermore, compound 2, but not 4, reduced elastase induced lung injury. Our data suggest that although coumarinic derivatives have anti-inflammatory properties, their anti-elastase activity is essential to reduce lung inflammation in vivo.
Asunto(s)
Antiinflamatorios , Cumarinas/farmacología , Macrófagos Alveolares/efectos de los fármacos , Neumonía/patología , Neumonía/prevención & control , Proteínas Inhibidoras de Proteinasas Secretoras , Animales , Línea Celular , Femenino , Interleucina-6/farmacología , Elastasa de Leucocito , Lipopolisacáridos , Pulmón/patología , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Neumonía/inducido químicamente , Ratas , Receptores CCR2/metabolismo , Escopoletina/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Pre-elafin is a tight-binding inhibitor of neutrophil elastase and myeloblastin; two enzymes thought to contribute to tissue damage in lung emphysema. Previous studies have established that pre-elafin is also an effective anti-inflammatory molecule. However, it is not clear whether both functions are linked to the antipeptidase activity of pre-elafin. As a first step toward elucidating the structure/function relationship of this protein, we describe here the construction and characterization of pre-elafin variants with attenuated antipeptidase potential. In these mutants, the P1' methionine residue of the inhibitory loop is replaced by either a lysine (pre-elafinM25K) or a glycine (pre-elafinM25G) residue. Both mutated variants are stable and display biochemical properties undistinguishable from WT (wild-type) pre-elafin. However, compared with WT pre-elafin, their inhibitory constants are increased by one to four orders of magnitude toward neutrophil elastase, myeloblastin and pancreatic elastase, depending on the variants and enzymes tested. As suggested by molecular modelling, this attenuated inhibitory potential correlates with decreased van der Waals interactions between the variants and the enzymes S1' subsite. In elastase-induced experimental emphysema in mice, only WT pre-elafin protected against tissue destruction, as assessed by the relative airspace enlargement measured using lung histopathological sections. Pre-elafin and both mutants prevented transient neutrophil alveolitis. However, even the modestly affected pre-elafinM25K mutant, as assayed in vitro with small synthetic substrates, was a poor inhibitor of the neutrophil elastase and myeloblastin elastolytic activity measured with insoluble elastin. We therefore conclude that full antipeptidase activity of pre-elafin is essential to protect against lung tissue lesions in this experimental model.
Asunto(s)
Elafina/genética , Elafina/metabolismo , Enfisema/patología , Pulmón/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Inhibidores de Serina Proteinasa/metabolismo , Animales , Elafina/química , Enfisema/metabolismo , Femenino , Regulación de la Expresión Génica , Cinética , Pulmón/citología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Neutrófilos/metabolismo , Conformación Proteica , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genéticaRESUMEN
The involvement of genes controlling embryonic processes in the etiology of diseases often escapes attention because of the focus given to their inherent developmental role. Hoxa5 belongs to the Hox gene family encoding transcription factors known for their role in skeletal patterning. Hoxa5 is required for embryonic respiratory tract morphogenesis. We now show that the loss of Hoxa5 function has severe repercussions on postnatal lung development. Hoxa5-/- lungs present an emphysema-like morphology because of impaired alveogenesis. Chronic inflammation characteristics, including goblet cell hyperplasia, mucus hypersecretion, and recruitment of inflammatory cells, were also observed. Altered cell specification during lung morphogenesis triggered goblet cell anomalies. In addition, the defective motility of alveolar myofibroblast precursors in the embryonic lung led to the mispositioning of the alveolar myofibroblasts and to abnormal elastin deposition postnatally. Both goblet cell hyperplasia and elastic fiber abnormalities contributed to the chronic physiopathological features of Hoxa5-/- lungs. They constituted an attractive stimulus to recruit activated macrophages that in turn generated a positive feedback loop that perpetuated macrophage accumulation in the lung. The present work corroborates the notion that altered Hox gene expression may predispose to lung pathologies.
Asunto(s)
Proteínas de Homeodominio/fisiología , Enfermedades Pulmonares/genética , Organogénesis/genética , Fosfoproteínas/fisiología , Alveolos Pulmonares/anomalías , Alveolos Pulmonares/crecimiento & desarrollo , Animales , Apoptosis/genética , Proliferación Celular , Elastina/análisis , Fibroblastos/química , Fibroblastos/citología , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/genética , Pulmón/anomalías , Pulmón/química , Pulmón/crecimiento & desarrollo , Ratones , Ratones Mutantes , Mutación , Tamaño de los Órganos/genética , Fosfoproteínas/análisis , Fosfoproteínas/genética , Alveolos Pulmonares/química , Células Madre/citología , Factores de TranscripciónRESUMEN
Few therapeutic options are offered to treat inflammation and alveolar wall destruction in emphysema. The effect of recombinant human pre-elafin, an elastase inhibitor, was evaluated in porcine pancreatic elastase (PPE)-induced emphysema in C57BL/6 mice. In a first protocol, mice received a single instillation of pre-elafin (17.5 pmol/mouse) at 1 h post-PPE and were sacrificed up to 72 h post-PPE. A single instillation of pre-elafin significantly reduced PPE-induced neutrophil accumulation in lungs, as assessed by bronchoalveolar lavage (BAL), by 51%, 71% and 67% at 24, 48 and 72 h, respectively. In a second protocol, mice also received a single dose of PPE, but pre-elafin three times a week for 2 weeks. After 2 weeks, pre-elafin significantly reduced the PPE-induced increase in BAL macrophage numbers, airspace dimensions and lung hysteresivity by 74%, 62% and 52%, respectively. Since G-CSF was previously shown to reduce emphysematous changes in mice, the BAL levels of this mediator were measured 6 h post-PPE in animals treated as described in the first protocol. Pre-elafin significantly increased G-CSF levels in PPE-exposed mice compared to sham- and PPE only-exposed animals. This suggests that the beneficial effects of pre-elafin could be mediated, at least in part, by its ability to increase G-CSF levels in the lung.
Asunto(s)
Enfisema/prevención & control , Factor Estimulante de Colonias de Granulocitos/metabolismo , Precursores de Proteínas/uso terapéutico , Animales , Líquido del Lavado Bronquioalveolar , Elafina , Enfisema/enzimología , Enfisema/metabolismo , Enfisema/fisiopatología , Femenino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismo , Proteínas Recombinantes/uso terapéuticoRESUMEN
BACKGROUND: Migration of eosinophils into bronchial mucosa requires proteolysis. Montelukast, a cysteinyl leukotriene (CysLT) 1 receptor antagonist used in asthma treatment, decreases eosinophil infiltration into the asthmatic airways, suggesting that CysLTs modulate eosinophil protease activity. OBJECTIVE: We sought to determine whether CysLTs and montelukast regulate eosinophil protease activity. METHODS: Purified blood eosinophils were treated with or without montelukast; MK-0591, a 5-lipoxygenase-activating protein inhibitor; or leukotriene (LT) D(4). Migration assays through Matrigel were performed in the presence of 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemotactic factor, or LTD(4). Expression of molecules implicated in plasmin generation and matrix metalloproteinase (MMP) 9 release were also evaluated. RESULTS: Montelukast and MK-0591 decreased eosinophil migration promoted by 5-oxo-ETE, whereas LTD(4) failed to induce eosinophil migration. However, LTD(4) significantly boosted the migration rate obtained with a suboptimal concentration of 5-oxo-ETE and partially reversed the inhibition obtained with MK-0591. Montelukast significantly reduced the maximal rate of activation of plasminogen into plasmin by eosinophils obtained with 5-oxo-ETE. 5-Oxo-ETE increased the number of eosinophils expressing urokinase plasminogen activator receptor and stimulated secretion of MMP-9. Montelukast, but neither MK-0591 nor LTD(4), reduced the expression of urokinase plasminogen activator receptor and the secretion of MMP-9 and increased total cellular activity of urokinase plasminogen activator and the expression of plasminogen activator inhibitor 2 mRNA. CONCLUSION: Montelukast inhibits eosinophil protease activity in vitro through a mechanism that might be independent of its antagonist effect on CysLT 1 receptor. CLINICAL IMPLICATIONS: This could partially explain montelukast's anti-inflammatory effect in asthma and eventually amplify to improve its therapeutic efficacy.
Asunto(s)
Acetatos/farmacología , Cisteína/fisiología , Eosinófilos/efectos de los fármacos , Antagonistas de Leucotrieno/farmacología , Leucotrienos/fisiología , Quinolinas/farmacología , Adulto , Ácidos Araquidónicos/farmacología , Movimiento Celular/efectos de los fármacos , Ciclopropanos , Activación Enzimática , Eosinófilos/enzimología , Eosinófilos/fisiología , Femenino , Humanos , Indoles/farmacología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Receptores de Superficie Celular/análisis , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Sulfuros , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Leukocytes and lung structural cells contribute to the pathophysiology of asthma through the production of numerous mediators including serine proteases. Such proteases include mast cell tryptase and chymase; neutrophil elastase, cathepsin G and myeloblastin (proteinase 3); bronchial epithelial cell-derived transmembrane protease, serine 11D (human airway trypsin-like protease); cytotoxic T lymphocyte- and natural killer cell-derived granzyme B; and, eosinophil serine protease 1 (testisin). Considerable effort to develop potent and selective inhibitors, mostly non-peptidic, especially targeting tryptase and chymase have been made in the last few years. This review presents promising inhibitors, currently in the research and development pipeline. Some endogenous inhibitors and other compounds purified from non-human species are also discussed.
Asunto(s)
Asma/tratamiento farmacológico , Inhibidores de Serina Proteinasa/uso terapéutico , Animales , Asma/enzimología , HumanosRESUMEN
The importance of early diagnosis to reduce the morbidity and mortality from cancer has led to a search for new sensitive and specific tumour markers. Molecular techniques developed over the past few years allow simultaneous screening of thousands of genes, and have been applied to different cancers to identify many genes that are modulated in various cancers. Of these, attention has focused on genes coding for a family of proteins with whey-acidic-protein (WAP) motifs. Most notably, the genes coding for elafin, antileukoproteinase 1 (previously called secretory leucocyte proteinase inhibitor, SLPI), WAP four disulphide core domain protein 1 (previously called prostate stromal protein 20 kDa, PS20), and WAP four disulphide core domain protein 2 (previously called major human epididymis-specific protein E4, HE4), have been identified as candidate molecular markers for several cancers. In this review, we assess data for an association between cancer and human WAP proteins, and discuss their potential role in tumour progression. We also propose a new mechanism by which WAP proteins might have a role in carcinogenesis.
Asunto(s)
Biomarcadores de Tumor/genética , Proteínas de la Leche/genética , Neoplasias/diagnóstico , Biomarcadores de Tumor/análisis , Proteínas Secretorias del Epidídimo/análisis , Proteínas Secretorias del Epidídimo/genética , Humanos , Proteínas de la Leche/análisis , Neoplasias/genética , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas/análisis , Proteínas/genética , Inhibidor Secretorio de Peptidasas Leucocitarias , beta-DefensinasRESUMEN
Circulating eosinophils need proteinases to mediate a spatially limited and orientated digestion of the extracellular matrix and to migrate into tissue. Moreover, proteinases are likely involved in tissue remodeling, a crucial feature of chronic diseases including asthma. Eosinophils express matrix metalloproteinase (MMP)-9, which is increased upon stimulation with TNF-alpha. Other MMPs, the membrane type (MT)-MMPs, likely play a major role in cell invasion and tissue remodeling. MT4-MMP was identified in peripheral blood leukocyte preparations, but it is not known whether eosinophils express MT4-MMP. We investigated the expression of MT4-MMP and its modulation by TNF-alpha in purified human blood eosinophils. The constitutive expression of MT4-MMP mRNA was detected by RT-PCR in unstimulated eosinophils, lymphocytes, and monocytes, but not neutrophils. Stimulation of eosinophils with TNF-alpha increased MT4-MMP mRNA expression. This effect appeared at 4h and reached a maximum at 8h of incubation. MT4-MMP protein was detected in freshly isolated blood eosinophils by Western blotting and immunocytochemistry. TNF-alpha increased expression of the MT4-MMP protein. MT4-MMP protein was also detected in nasal polyp eosinophils by immunohistochemistry. In conclusion, eosinophils constitutively express MT4-MMP, which is increased upon stimulation with TNF-alpha. Consequently, MT4-MMP may be directly involved in the degradation of extracellular matrix components and/or modulate the activity of other proteins implicated in eosinophil migration and tissue remodeling.
Asunto(s)
Eosinófilos/enzimología , Metaloproteinasas de la Matriz , Metaloendopeptidasas/metabolismo , Células Cultivadas , Eosinófilos/citología , Eosinófilos/efectos de los fármacos , Humanos , Inmunohistoquímica , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Pólipos Nasales/metabolismo , Pólipos Nasales/patología , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Neutrophil elastase is a protease that is involved in the tissue destruction and inflammation that characterize numerous diseases, including hereditary emphysema, chronic obstructive pulmonary disease, cystic fibrosis, adult respiratory distress syndrome, ischemic-reperfusion injury and rheumatoid arthritis. Thus, elastase has been the object of extensive research to develop potent inhibitors that target its destructive and pro-inflammatory action. This review focuses on the anti-inflammatory activity of inhibitors that are currently, or were until recently in development, including purified or recombinantly produced endogenous inhibitors, genetically modified recombinant protein inhibitors and synthetic small-molecule inhibitors.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Elastasa de Leucocito/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/química , Humanos , Elastasa de Leucocito/metabolismo , Serpinas/química , Serpinas/farmacología , Serpinas/uso terapéuticoRESUMEN
The aim of the present study was to evaluate the anti-inflammatory activity of pre-elafin, an elastase-specific inhibitor, in lipopolysaccharide (LPS)-induced acute lung inflammation. C57BL/6 mice were pre-treated intranasally with recombinant human pre-elafin or vehicle only. One hour later, they were instilled intranasally with LPS (2 microg/mouse). Animals were sacrificed 6 hours after LPS instillation and bronchoalveolar lavage (BAL) was performed with three 1-ml aliquots of saline. LPS induced a lung inflammation characterised by a 100-fold increase in BAL neutrophils compared to control animals (265.8 +/- 54.5 x 10(3) and 2.4 +/- 1.3 x 10(3) neutrophils/ml, respectively). Pre-elafin dose-dependently reduced the neutrophil influx in the lung alveolar spaces by up to 84%. No elastase activity was detectable in all BAL fluids tested. Pre-elafin also reduced significantly LPS-induced gelatinase activity, as shown by zymography, and BAL macrophage inflammatory protein-2 (MIP-2) and KC levels, two potent neutrophil attractants and activators. Moreover, pre-elafin also significantly reduced mRNA levels of the three members of the IL-1 ligand family, namely IL-1alpha, IL-1beta and IL-1 receptor antagonist (IL-1Ra), type II IL-1 receptor, and TNFalpha as assessed in whole lung tissue by RNase protection assay. Thus, pre-elafin may be considered as a potent anti-inflammatory mediator.
Asunto(s)
Antiinflamatorios/farmacología , Precursores Enzimáticos/farmacología , Neumonía/tratamiento farmacológico , Proteínas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Enfermedad Aguda , Animales , Líquido del Lavado Bronquioalveolar/citología , Citocinas/efectos de los fármacos , Femenino , Gelatinasas/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Elastasa Pancreática/efectos de los fármacos , Neumonía/inducido químicamente , Neumonía/inmunología , Proteínas Inhibidoras de Proteinasas SecretorasRESUMEN
Asthma is characterized by an airway remodeling process involving altered extracellular matrix deposition such as collagen, fibronectin and proteoglycans. Proteoglycans determine tissue mechanical properties and are involved in many important biological aspects. Not surprisingly, it has been suggested that proteoglycan deposition may alter airway properties in asthma including airway hyperresponsiveness. In chronically inflamed airway tissues, fibroblasts likely represent an activated fibrotic phenotype that contributes to the excessive deposition of different extracellular matrix components. To investigate whether this was the case for proteoglycans, the production of hyaluronan, perlecan, versican, small heparan sulphate proteoglycans (HSPGs), decorin and biglycan was quantified in the culture medium of primary bronchial fibroblast cultures, established from four normal and six asthmatic subjects. Values were further correlated to the airway responsiveness (PC(20) methacholine) of donor subjects. Fibroblasts from subjects with the most hyperresponsive airways produced up to four times more total proteoglycans than cells from subjects with less hyperresponsive or normoresponsive airways. We observed a significant negative correlation between the PC(20) and perlecan, small HSPGs and biglycan, while such correlation was absent for decorin and close to significant for hyaluronan and versican. Altered proteoglycan metabolism by bronchial fibroblasts may contribute to the increased proteoglycan deposition in the bronchial mucosa and to airway hyperresponsiveness characterizing asthma.
Asunto(s)
Asma/metabolismo , Bronquios/fisiopatología , Proteoglicanos/biosíntesis , Asma/fisiopatología , Western Blotting , Bronquios/citología , Bronquios/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Cromatografía Liquida , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Ácido Hialurónico/biosíntesis , Técnicas In VitroRESUMEN
STUDY OBJECTIVES: Pre-elafin, also known as trappin-2, is an elastase-specific inhibitor that could be an ideal candidate for the treatment of neutrophil elastase-driven lung diseases. The inhibitory activity of pre-elafin resides in the COOH-terminal region that can be released as mature elafin. The NH(2)-terminal moiety of pre-elafin is characterized by the presence of a specific repeating sequence, termed cementoin, believed to immobilize the inhibitor to lung protein components and restrict its diffusion from the desired sites of action. This property should confer an advantage to pre-elafin compared to elafin in the treatment of neutrophil elastase-driven lung diseases. MEASUREMENTS: The inhibitory effect of recombinant human pre-elafin was assessed in a human neutrophil elastase-induced acute lung injury model in Golden Syrian hamsters. BAL fluid hemoglobin content was used as a marker of lung injury. RESULTS: Recombinant human pre-elafin administered intratracheally 1 h prior to neutrophil elastase dose-dependently inhibited the lung hemorrhage with a calculated half-effective dose of 8.1 microg/kg (0.7 nmol/kg). Pre-elafin was equally efficient when administered 3 h before neutrophil elastase. In contrast to pre-elafin, commercial synthetic elafin was ineffective in inhibiting neutrophil elastase-induced lung hemorrhage even at a dose of 4.45 nmol/kg. CONCLUSIONS: Our results suggest that pre-elafin may be eventually used in the treatment of neutrophil elastase-driven lung diseases.