RESUMO
Cigarette smoking is the major risk factor for the development of pulmonary emphysema, a disorder that may result from an imbalance between the elastase and antielastase levels in the lungs. Decreased functional alpha 1-protease inhibitor, an inhibitor of neutrophil elastase, might render smokers susceptible to elastase-catalyzed destruction of pulmonary elastic fibers and the development of emphysema. Binding and inactivation of isotopically labeled porcine pancreatic elastase and human neutrophil elastase by alpha 1-protease inhibitor were measured in fluid obtained by bronchoalveolar lavage of volunteers. The inhibition of elastase-catalyzed solubilization of elastin and a tripeptide substrate were also determined. The mean level of functional alpha 1-protease inhibitor in the bronchoalveolar lavage fluid of smokers was found to be equal to or greater than that of nonsmokers, contradicting reports by other investigators. Increased elastase derived from pulmonary neutrophils, rather than decreased functional alpha 1-protease inhibitor, appears to be the main factor in the genesis of emphysema in smokers.
Assuntos
Brônquios/metabolismo , Inibidores de Proteases/metabolismo , Alvéolos Pulmonares/metabolismo , Fumar , Adulto , Feminino , Humanos , Técnicas In Vitro , Masculino , Neutrófilos/metabolismoRESUMO
Desmosine (DES) and isodesmosine (IDES) are two unusual, tetrafunctional, pyridinium ring-containing amino acids involved in elastin cross-linking. Being amino acids unique to mature, cross-linked elastin, they are useful for discriminating peptides derived from elastin breakdown from precursor elastin peptides. According to these features, DES and IDES have been extensively discussed as potentially attractive indicators of elevated lung elastic fibre turnover and markers of the effectiveness of agents with the potential to reduce elastin breakdown. In the present manuscript, immunology-based and separation methods for the evaluation of DES and IDES are discussed, along with studies reporting increased levels of urine excretion in chronic obstructive pulmonary disease (COPD) patients with and without alpha(1)-antitrypsin deficiency. The results of the application of DES and IDES as surrogate end-points in early clinical trials in COPD are also reported. Finally, recent advances in detection techniques, including liquid chromatography tandem mass spectrometry and high-performance capillary electrophoresis with laser-induced fluorescence, are discussed. These techniques allow detection of DES and IDES at very low concentration in body fluids other than urine, such as plasma or sputum, and will help the understanding of whether DES and IDES are potentially useful in monitoring therapeutic intervention in COPD.
Assuntos
Desmosina/sangue , Elastina/metabolismo , Doença Pulmonar Obstrutiva Crônica/sangue , Adulto , Criança , Cromatografia Líquida/métodos , Feminino , Humanos , Isodesmosina/sangue , Masculino , Modelos Biológicos , Peptídeos/química , Fumar , Espectrometria de Massas em Tandem/métodos , Deficiência de alfa 1-Antitripsina/sangueRESUMO
Radiolabeled, enzymatically active or chloromethyl ketone-inactivated porcine pancreatic elastase was endotracheally instilled into hamsters. Gel filtration of the bronchopulmonary lavage fluid revealed two major radioactive fractions: one, eluting at 780,000 daltons, corresponding to an alpha-macroglobulin-pancreatic elastase complex, and another, at 68,000 daltons, corresponding to an alpha-1-protease inhibitor-pancreatic elastase complex. Elastolytic activity was recovered in the bronchopulmonary lavage fluid up to 4 d after elastase instillation and was associated with the alpha-macroglobulin-pancreatic elastase complex. Small amounts of this complex were recovered 14 d after instillation. When less than 1% (1.5--1.7 micrograms) of the usual dose of elastase was instilled into hamsters, the major radioactive complex was alpha-1-protease inhibitor-pancreatic elastase complex, and little or no elastolytic activity was found in the lavage fluid. In contrast to the instillation of 220 micrograms of elastase, no disease or hemorrhagic reaction was detected with this low dose, and without hemorrhage only insignificant amounts of alpha-macroglobulin-pancreatic elastase complexes were recovered from the lungs. To study the interaction of circulating antiproteases with elastase, hamster plasma was allowed to interact directly with the radiolabeled elastase; alpha-macroglobulin bound much more of the elastase than alpha-1-protease inhibitor, confirming the findings in the lung lavage experiments. The hamster's susceptibility to pancreatic elastase-induced emphysema may depend on the preferential binding of elastase to alpha-macroglobulin, which protects the elastolytic potential, rather than to alpha-1-protease inhibitor, which inactivates elastase. We speculate that if even a fraction of the residual radioactivity found in the hamster lungs as long as 144 d after instillation of elastase represents enzymatically active alpha-macroglobulin-pancreatic elastase complex, this could serve as a source of persistent elastolytic activity, which might explain the progressive nature of the pulmonary lesion.
Assuntos
Pulmão/metabolismo , Elastase Pancreática/metabolismo , Enfisema Pulmonar/etiologia , alfa-Macroglobulinas/metabolismo , Animais , Cromatografia em Gel , Cricetinae , Masculino , Mesocricetus/metabolismo , Elastase Pancreática/efeitos adversos , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/metabolismo , Fatores de TempoRESUMO
The objective of this study was to investigate the elastin repair process in the rat aortic smooth muscle cell culture after proteolytic injury. Although little studied in vivo, elastin repair is thought to occur through a sequential process involving enzymatic removal (debridement) of damaged fibers followed by synthesis of tropoelastin, its subsequent processing, and eventual incorporation into new insoluble elastin. A second repair mechanism of proteolytically damaged elastin in a culture system is reported here. Repair in this system relates directly to restoration of resistance to elastin solubilization by hot alkali. As expected, severe injuries were observed with porcine pancreatic elastase (PPE). Using PPE, only 6% of the elastin, relative to control, was resistant to hot alkali immediately after elastase treatment. 4 wk later, resistance to hot alkali had increased dramatically to a mean of 90%. Repair took longer after injury with 75 micrograms of PPE as compared with 50 micrograms of PPE. The limited elastic fiber proteolysis induced by either human neutrophil elastase or porcine trypsin was repaired in culture within 2 wk. Elastin that had been radiolabeled with [3H]lysine 4-5 wk before injury was converted from a hot NaOH-susceptible to a NaOH-resistant elastin fraction during recovery from PPE injury. At the same time, the frayed elastic fibers that were seen with the electron microscope immediately after PPE treatment were replaced by continuous bands of elastin that resembled those in control cultures. Restoration of NaOH resistance did not require a net increase in total cell layer elastin, suggesting that relatively little new tropoelastin incorporation into the cell layer was required for this type of repair. These results suggested a salvage repair mechanism for proteolytically damaged elastin.
Assuntos
Elastina/metabolismo , Músculo Liso Vascular/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Elastina/ultraestrutura , Concentração de Íons de Hidrogênio , Lisina/farmacocinética , Microscopia Eletrônica , Ratos , Ratos Endogâmicos , Hidróxido de Sódio/farmacologia , SolubilidadeRESUMO
The neutrophil serine proteinases elastase and cathepsin G produce connective tissue injury, the extent of which depends on the balance between these enzymes and their inhibitors. The most important of these inhibitors is alpha 1-proteinase inhibitor, a member of a superfamily of homologous proteins known as serpins. Neutrophil cytosol inhibited the activities of human neutrophil elastase and cathepsin G in a dose-dependent fashion. To demonstrate formation of an enzyme-inhibitor complex, we combined 125I-elastase or 125I-cathepsin G with neutrophil cytosol or alpha 1-proteinase inhibitor and analyzed the products by polyacrylamide gel electrophoresis. Unbound elastase and cathepsin G each migrated to an apparent molecular weight of 25 kDa. In the presence of cytosol from neutrophils both radiolabeled enzymes migrated with a relative size of 68 kDa, whereas in the presence of alpha 1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100 degrees C but were dissociated by hydrolysis in ammonium hydroxide (1.5 mol/L) at 37 degrees C. Formation of each complex was prevented by pretreatment of elastase or cathepsin G with diisopropylfluorophosphate, indicating that the inhibitor binds to the active site of the enzyme. Exposure of either alpha 1-proteinase inhibitor or neutrophil cytosol to the myeloperoxidase-H2O2-halide system prevented complex formation, suggesting the presence of an oxidizable amino acid at the binding site of the inhibitor. By electrophoretic analysis, the molecular weight of the cytosolic inhibitor was 43 kDa and neutrophils contained approximately 1 attomol of inhibitor per cell. The isoelectric points of the elastase and cathepsin G inhibitor were 5.5-5.9 and inhibitors of the two proteinases coeluted using size exclusion chromatography. These data demonstrate that human neutrophil cytosol contains a single serpinlike protein that inhibits elastase and cathepsin G. The inhibitor may be important in protecting the intracellular environment from proteolytic injury during degranulation.
Assuntos
Catepsinas/antagonistas & inibidores , Neutrófilos/enzimologia , Elastase Pancreática/antagonistas & inibidores , Inibidores de Proteases/metabolismo , Hidróxido de Amônia , Catepsina G , Citosol/enzimologia , Humanos , Peróxido de Hidrogênio/metabolismo , Hidróxidos/química , Técnicas In Vitro , Ponto Isoelétrico , Peso Molecular , Oxirredução , Peroxidase/metabolismo , Inibidores de Proteases/química , Serina Endopeptidases , Células Tumorais CultivadasRESUMO
Improved conditions were used for the aseptic growth of Arabidopsis thaliana to investigate whether xylem colonization of A. thaliana by Azorhizobium caulinodans ORS571 might occur. When seedlings were inoculated with ORS571 (pXLGD4) tagged with the lacZ reporter gene, nearly all of the plants showed blue regions of ORS571 colonization at lateral root cracks (LRC). The flavonoids naringenin and liquiritigenin significantly stimulated colonization of LRC by ORS571. Blue bands of ORS571 (pXLGD4) bacteria were observed histochemically in the xylem of intact roots of inoculated plants. Detailed microscopic analysis of sections of primary and lateral roots from inoculated A. thaliana confirmed xylem colonization. Xylem colonization also occurred with an ORS571 nodC mutant deficient in nodulation factors. There was no significant difference in the percentage of plants with xylem colonization or in the mean length of xylem colonized per plant between plants inoculated with either ORS571 (pXLGD4) or ORS571::nodC (pXLGD4), with or without naringenin.
Assuntos
Arabidopsis/microbiologia , Azorhizobium caulinodans/crescimento & desenvolvimento , Arabidopsis/metabolismo , Azorhizobium caulinodans/genética , Azorhizobium caulinodans/patogenicidade , Genes Reporter , Óperon Lac , Fixação de Nitrogênio , Raízes de Plantas/microbiologiaRESUMO
We used ultrastructural and immunocytochemical technique to follow the movement of human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) through a living extracellular matrix produced by cultured smooth muscle cells and to compare the effect of the two elastases on elastic fibers in situ. Although both enzymes solubilize elastin purified from these cultures at similar rates, PPE solubilized 11.5 times more elastin from the intact cultures than did HNE. The difference in the rate of elastin solubilization from the cultures parallels the degree of elastic fiber degradation and the emphysema-inducing potency of the two elastases when they are instilled into animal lungs. Immunohistochemical studies employing antibodies to HNE and PPE revealed that PPE penetrates the smooth muscle cell cultures more readily than does HNE. Because the amount of elastin in these cultures increases with increasing distance from the free surface, the lesser amounts of elastin solubilized by HNE may be partly due to poor penetration of HNE into the living extracellular matrix, resulting in limited access to elastin substrate. Ultrastructural and immunocytochemical studies indicated, however, that even when HNE does have access to elastin substrate, it is less efficient than PPE at penetrating and degrading individual elastic fibers.
Assuntos
Aorta/citologia , Elastina/análise , Proteínas da Matriz Extracelular/análise , Matriz Extracelular/ultraestrutura , Músculo Liso Vascular/citologia , Elastase Pancreática/metabolismo , Animais , Animais Recém-Nascidos , Aorta/ultraestrutura , Células Cultivadas , Humanos , Imuno-Histoquímica/métodos , Elastase de Leucócito , Microscopia Imunoeletrônica/métodos , Músculo Liso Vascular/ultraestrutura , Neutrófilos/enzimologia , Elastase Pancreática/análise , Coelhos/imunologia , Ratos , SuínosRESUMO
Much of the experimental evidence supporting the hypothesis that pulmonary emphysema results from an imbalance between elastases and anti-elastases in the lung comes from animal models. The present study was designed to examine the effects on human lung tissue of the two elastases that have been most widely used to produce these animal models. Lung tissue was exposed in vitro to human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE). Although both enzymes solubilized protein at similar rates, PPE solubilized elastin five times faster than did HNE. Ultrastructurally, HNE-exposed tissue exhibited fewer damaged elastic fibers as well as some fibers that were damaged at the edges, whereas the interior of the fiber appeared intact. Elastic fibers showing damage only at the periphery were not seen in tissue exposed to PPE. Immunocytochemical studies in which antibodies to HNE and PPE were applied to thin sections of Lowicryl-embedded tissue indicated that both of these elastases could be detected in association with elastic fibers, but only in areas of the fiber that showed morphological evidence of elastase injury. Both HNE and PPE removed fibronectin from basement membranes (as determined by loss of binding of fibronectin antibodies after exposure to elastase), but neither elastase was detected on basement membrane. Loss of epithelial cells usually accompanied elastic fiber damage by HNE but not PPE.
Assuntos
Pulmão/ultraestrutura , Elastase Pancreática/fisiologia , Animais , Enfisema/enzimologia , Humanos , Imuno-Histoquímica , Pulmão/metabolismo , Pulmão/fisiopatologia , Microscopia Eletrônica , SuínosRESUMO
Palladium chloride in aqueous solution stains elastic fibers in thin sections of Epon-embedded tissues. When palladium chloride is used with a lead citrate counterstain, high contrast sections with gray to black elastic fibers are obtained. The stain was tested on newborn and adult mammalian tissues and on adult tissues from lower animals. Sections were mounted on stainless steel grids, stained with 1% palladium chloride solution for 5 to 15 min, rinsed thoroughly, and counterstained with lead citrate for 7 min. Palladium chloride staining solution is stable for several months at room temperature and if the stain is filtered immediately before use, contamination of sections is not a problem. Chemical studies indicate that palladium binds directly to purified bovine ligamentum nuchae elastin and that this binding is not affected by glutaraldehyde fixation or by sodium borohydride reduction of elastin. Osmium post-fixation of glutaraldehyde-fixed elastin did significantly lower the amount of palladium bound. Palladium was shown to be chemically bound to sites on the elastin and not weakly associated. The nature of these sites is discussed.
Assuntos
Elastina/análise , Paládio , Coloração e Rotulagem/métodos , Sacos Aéreos/ultraestrutura , Animais , Aorta/ultraestrutura , Bovinos , Peixes , Ligamentos/ultraestrutura , Pulmão/ultraestrutura , Camundongos , Artéria Pulmonar/ultraestrutura , TartarugasRESUMO
Emphysema in humans takes several different forms: centrilobular, panacinar, paraseptal, and airspace enlargement with fibrosis. The varying morphologic and background features of these forms of emphysema suggest that they differ in pathogenesis. Elastic fiber rupture and fraying are a feature of emphysema. Experimental emphysema may be induced by human neutrophil elastase and other elastolytic enzymes but not by nonelastolytic proteases. Disruption of elastic fibers also appears to be the underlying feature of lathyrogen-induced airspace enlargement and of the emphysema in the blotchy mouse. However, there is no evidence of elastic fiber destruction in cadmium-induced airspace enlargement with fibrosis or in emphysema associated with hyperoxia or severe starvation. Thus, elastic fiber disruption is not common to all forms of experimental emphysema. We posit that airspace enlargement may be a stereotyped response of the lungs to different injuries. Emphysema can be induced in experimental animals by repeated induction of pulmonary neutrophilia. However, the evidence for involvement of neutrophil elastase in human emphysema is not clear: there are studies using a variety of approaches that weigh on both sides of the question. There is also in vitro evidence that alveolar macrophages can degrade elastin or elastic fibers with which they are in contact by means of a metalloelastase or the cooperative action of plasminogen activator and an acid cysteine protease. We conclude that the pathogenesis of emphysema is complex. Neutrophil elastase likely plays a major role in the development of some forms of emphysema, but our understanding of the interactions between the alveolar walls and neutrophils is still fragmentary.
Assuntos
Elastase Pancreática/fisiologia , Enfisema Pulmonar/etiologia , Animais , Cádmio/toxicidade , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Elastase de Leucócito , Pulmão/enzimologia , Neutrófilos/enzimologia , Elastase Pancreática/análiseRESUMO
Liver fibrosis is a serious complication of schistosomiasis infection, is associated with increased amounts of collagen and the collagen cross-link, pyridinoline. Non-invasive markers of liver fibrosis have been developed. Serum and urinary markers of collagen synthesis and degradation have been studied to assess the balance between collagen synthesis, measured with markers of collagen synthesis such as amino-terminal propeptide of type III procollagen (PIIINP), and markers of degradation such as pyridinoline or pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP). It has been shown that mice infected with Schistosomiasis mansoni excrete excess pyridinoline cross links in urine and this was correlated with the collagen content of granulomas from the liver. Treatment of infected mice with an anti-parasitic drug, praziquantel, decreased the collagen content of parenchyma and excretion of pyridinoline in the urine. Although the connective tissue protein, elastin, is present in the liver, the role of elastin in liver fibrosis has not been investigated. However, it has been shown that the urinary concentration of elastin specific crosslinks, desmosine and isodesmosine, as well as the urinary concentration of the collagen crosslink, pyridinoline, correlated well with liver fibrosis score in biopsy specimens from patients with liver disease secondary to hepatitis C virus and alcohol. Each biopsy specimen was reviewed by two pathologists who were blinded as to the clinical data. The pathological evaluation generated scores for both inflammation and fibrosis. No correlation was seen between the urinary markers and inflammation scores. The measurement of non-invasive markers of collagen synthesis and degradation may be useful in monitoring the reversal of fibrosis following therapeutic intervention in schistosome infections.
Assuntos
Colágeno/metabolismo , Elastina/metabolismo , Cirrose Hepática/metabolismo , Esquistossomose/complicações , Aminoácidos/sangue , Aminoácidos/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Desmosina/urina , Humanos , Isodesmosina/urina , Cirrose Hepática/sangue , Cirrose Hepática/etiologia , Cirrose Hepática/urinaRESUMO
Native pancreatic elastase and guanidinated elastase have similar in vitro and in vivo properties and produce emphysema of similar severity in hamsters. 14C-guanidinated pancreatic elastase (16,000 cpm/0.2 mg) was instilled into the trachea of anesthetized hamsters. Within 24 hours the radiolabel found in the lungs rapidly decreases to 12% of the original 16,000 cpm and to 1% after 96 hours. Most of the radiolabel and elastase activity found in the lungs can be removed by bronchopulmonary lavage up to 48 hours after installation. Although seemingly very small, there is a significant amount of radiolabel (1-2%) which cannot be removed from the lungs by extensive bronchopulmonary lavage.