A lipophilic heparin derivative protects elastin against degradation by leucocyte elastase.

An oleolylated derivative (I) of partially N-desulphated heparin was prepared containing an average number of three oleoyl residues for one molecule of heparin. The inhibitory capacity of I (IC50 = 0.55 microM) for leucocyte elastase resembles that of heparin (IC50 = 0.2 microM). In contrast to heparin, I is also an inhibitor of porcine pancreatic elastase (IC50 = 0.68 microM) and it also has the capacity to protect elastin fibres against the degradation by leucocyte elastase. When insoluble elastin is pretreated with I its degradation by leucocyte elastase is inhibited by almost 90% while pretreatment of elastin with heparin exhibited only a moderate effect on elastolysis (10% inhibition).     

The identification and estimation of elastase in serum and plasma

1. Electrophoretic separation of partially purified elastase preparations from pancreas followed by incubation of the electrophoretogram in contact with an agar gel containing 4% of either Congo Red-stained or unstained elastin demonstrated that the enzyme which dissolves elastin can be identified with that which releases dye from the stained preparation. 2. A method for the estimation of elastase based on the release of dye from Congo Red-stained elastin is described. It is 23 times as sensitive as methods employing protein determination. 3. With the method, elastase activity can be identified in plasma and in a partially fractionated plasma protein preparation. 4. Lineweaver–Burk plots of these estimates of activity at a variety of substrate concentrations indicate that the reaction between elastase and the dyed elastin is more closely similar to that between elastase and the soluble substrate elastin rather than to that between elastase and the solid substrate. 5. Values for K_m and V_max. calculated for the enzyme present in plasma present further evidence for its identity with the pancreatic enzyme. 6. By calculation of the slopes of the Lineweaver–Burk plots for various enzyme concentrations it has proved possible to demonstrate that the inhibitor which is also present in the plasma is without effect on the enzyme when it acts on the dyed substrate.     

The degradation of human lung elastin by neutrophil proteinases

Human lung elastin has been isolated by both a degradative and nondegradative procedure and the products obtained found to have amino acid compositions comparable to published results. These elastin preparations, when utilized as substrates for various mammalian proteinases, were solubilized by porcine elastase at a rate six times faster than human leukocyte elastase. Leukocyte cathepsin G also solubilized lung elastin but only at 12% of the rate of the leukocyte elastase. In all cases the elastin prepared by nondegradative techniques proved to be the best substrate in these studies. The differences in the rate of digestion of elastin of the two elastolytic proteinases was readily attributed to the specificity differences of each enzyme as judged by carboxyterminal analysis of solubilized elastin peptides. The plasma proteinase inhibitors, alpha-1-proteinase inhibitor and alpha-2-macroglobulin abolished the elastolytic activity of both leukocyte enzymes, while alpha-1-antichymotrypsin specifically inactivated cathespsin G. Two synthetic inhibitors, Me-O-Suc-Ala-Ala-Pro-Val-CH2Cl (for elastase and Z-Gly-Leu-Phe-CH2Cl (for cathepsin G) were equally effective in abolishing the elastolytic activity of the two neutrophil enzymes. However, inhibition of leukocyte elastase by alpha-1-proteinase inhibitor was significantly suppressed if the enzyme was preincubated with elastin prior to addition of the inhibitor.     

Degradation of galactomannan by aClostridium butyricum strain

During staphylococcal pneumonia massive destruction of lung tissue is often observed. Staphylococcal serine proteinase (SSP) inactivates -1-proteinase inhibitor (1PI) a major factor which protects lungs from phagocyte proteases. We investigated the effect of SSP on elastin degradation by porcine pancreatic elastase (PE) and crude extract of human neutrophil elastase (NE) in solution and gel containing 1PI. SSP having no elastase activity enhanced PE and NE-induced elastinolysis in solution when added to 1PI before mixing with elastase and then with elastin. SSP added simultaneously with 1PI to PE had no influence on elastin degradation. However, SSP added simultaneously, 30 min before or 30 min after PE significantly increased elastin digestion in elastin-agarose plate with 1PI. Maximal increase in elastinolysis about 3-fold was for SSP added 30 min prior to PE. Since elastin is the major component of the alveolar walls it is possible that lung damage in the course of staphylococcal infection may partly depend on action of SSP.Abbreviations 1PI -1-proteinase inhibitor - NE crude extract of human neutrophil elastase - PE porcine pancreatic elastase - PBS phosphate buffered saline - SSP staphylococcal serine proteinase     

Pulmonary fibroblasts: an in vitro model of emphysema. Regulation of elastin gene expression

Disruption and degradation of interstitial elastic fibers are significant characteristics of pulmonary emphysema. In order to examine the responses of elastogenic cells to the conditions mimicking degradation of interstitial pulmonary elastin, rat pulmonary fibroblast cultures were used as an in vitro model. Second passage fibroblasts were divided into two different environmental situations to represent cells adjacent to and remote from the site of elastase-digested matrix. One set of cell cultures was briefly digested with pancreatic elastase. The resultant digest was then added back incrementally to the medium of elastase-digested cell cultures and to the medium of a second set of undigested cultures. Both sets of cell cultures remained viable and metabolically active during these treatments (96 h of incubation) as judged by protein synthesis, cell number, and steady-state levels of beta-actin mRNA. However, the two sets of cultures exhibited opposite responses in elastin gene expression with addition of increasing amounts of the elastase digest. The elastase-digested cultures exhibited a 200% increase in extractable soluble elastin and a 186% increase in tropoelastin mRNA with the addition of increasing amounts of the elastase digest to the medium. Conversely, the amount of soluble elastin recovered from the undigested cultures decreased 75%, and the steady-state level of tropoelastin mRNA decreased 63%. Soluble elastin peptides generated from oxalic acid treatment of purified elastin were shown to decrease tropoelastin mRNA in undigested cell cultures in the same manner as the elastase digest. Based on these data, we propose that pulmonary fibroblast elastin gene expression can be controlled coordinately by the state of the extracellular matrix and solubilized peptides derived from that matrix. Such integrated regulation may serve to localize elastin repair mechanisms.     

Primary enzyme quantitation using substrates labeled with a second indicator enzyme. I. Elastase determination using peroxidase-labeled elastin

Quantitation of proteolytic enzyme concentration can be accomplished by measuring the release, due to primary enzyme catalysis, of a second enzyme bound to a particulate substrate. As the primary enzyme acts on the substrate, release of the indicator enzyme into the surrounding medium occurs, which in turn can be quantitated colorimetrically, and under suitable reaction conditions the amount of indicator enzyme released is directly proportional to the amount of primary enzyme present. A specific example of such an assay is that for elastolytic activity using powdered elastin labeled with horseradish peroxidase. The detection sensitivity of the system described is 1 ng/ml of pancreatic elastase, and the dynamic range of the assay is 2 orders of magnitude. The reaction time for optimal elastase detection sensitivity is 3 h. For the assay, horseradish peroxidase is coupled to insoluble elastin. Labeled elastin is incubated with varying amounts of pancreatic elastase. The elastase in the test sample solubilizes the elastin and the horseradish peroxidase bound to it. The amount of peroxidase released is then quantified using the colorimetic reaction produced by catalysis of 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonate)-H₂O₂. For a fixed, nonsaturating concentration of elastase, the amount of peroxidase released is proportional to the elastase concentration.     

Change in elastin structure in human aortic connective tissue diseases

Summary Biochemical pathogenesis of the aortic connective tissue diseases (such as, Marfan's syndrome, dissecting aneurysm or aortic aneurysm) was examined by estimating glycoprotein, collagen and elastin contents in the aorta and the intramolecular cross-linking component (isodesmosine) and the intermolecular cross-linking components (cystine, histidinoalanine) in comparison with the control samples obtained from subjects with aortic regurgitation. The elastin content in the aorta and isodesmosine content obtained from the extract of the aortic sample found to be decreased. Ratio of cysteine residues (Cys/Cys-Cys) in the elastin fraction in disease increased. Content of histidinoalanine was found to be decreased. It may be suggested that elastin is maintained in its native nature and shape by intra- and inter-molecular cross-linking bridges, and they are readily denatured by various disease conditions. After elastin was solubilized by elastase, immunoreactive elastin content in those aortic diseases was found to be increased in the human connective tissue. Serum elastase and elastase-like activities tend to increase more than those in the control. These findings may suggest that the change in the structure of elastin would make more susceptible to elastase and other proteolytic enzymes. The reasonable hypothesis may be that molecular defect of fibillin or other constitutional structural glycoproteins produce deficient and functionally incompetent elastin associated microfibrils, and the defect of microfibrils cause to insufficient intra- and inter-molecular cross-links in elastin.     

The electron microscopic immunohistochemistry of elastase-treated aorta and nuchal ligament of fetal and postnatal sheep

In conjunction with the immunoperoxidase and the immunoferritin methods, antielastin antibody was used to study the localization of elastin in untreated and elastase-treated elastic fibers of the nuchal ligament and the aorta of fetal and young adult sheep. In tissues not treated with elastase, the staining reaction for antielastin antibody was localized in the outer zones of the amorphous components and along the surfaces of the microfibrils ; the central zones of the amorphous components were unreactive. After mild elastase treatment, incompletely digested amorphous components showed staining both in their central and outer zones, and some of the microfibrils became unreactive. After extensive elastase treatment, small scattered amorphous components were still found in association with bundles of microfibrils. These components were stained diffusely by the antielastin antibody method but were not detectable by staining with uranyl acetate and lead citrate or with Kajikawa 's method for elastin; elastin was not detected on the surfaces of the microfibrils by any of the methods used. These findings were interpreted as indicating that the surfaces of the microfibrils are associated with small amounts of elastin, and that evenly stained amorphous components are composed of elastin, which is loosely arranged and allows the penetration of antielastin antibody. These observations support the concept that microfibrils serve an important role as a scaffold for elastin deposition in elastogenesis. Because of their high sensitivity, immunohistochemical methods for detecting elastin are useful to study partially degraded elastic fibers.     

Liberation of desmosine and isodesmosine as amino acids from insoluble elastin by elastolytic proteases

The development of atherosclerotic lesions and abdominal aortic aneurysms involves degradation and loss of extracellular matrix components, such as collagen and elastin. Releases of the elastin cross-links desmosine (DES) and isodesmosine (IDE) may reflect elastin degradation in cardiovascular diseases. This study investigated the production of soluble elastin cross-linking structures by proteinases implicated in arterial diseases. Recombinant MMP-12 and neutrophil elastase liberated DES and IDE as amino acids from insoluble elastin. DES and IDE were also released from insoluble elastin exposed to monocyte/macrophage cell lines or human primary macrophages derived from peripheral blood monocytes. Elastin oxidized by reactive oxygen species (ROS) liberated more unconjugated DES and IDE than did non-oxidized elastin when incubated with MMP-12 or neutrophil elastase. These results support the exploration of free DES and IDE as biomarkers of elastin degradation.     

A Versatile Microassay for Elastase Using Succinylated Elastin

We have developed a rapid, versatile, and sensitive elastase assay that is based on the measurement of primary amines that are exposed due to enzymatic degradation of proteins, using succinylated elastin as the substrate for elastase. After incubation with elastase the degree of digestion is determined with trinitrobenzene sulfonic acid. The assay is rapid and sensitive, detecting elastase down to 1 ng/ml, and is linear up to enzyme concentrations of 10 μg/ml. The assay is carried out in microtiter plate wells and therefore offers the potential for assaying numerous samples of small volume. The use of succinylated elastin shows specificity for elastase over the control protease, trypsin. This assay is also versatile because it can be applied to samples such as cell culture supernatants, blood plasma, tissue biopsies, and tissue homogenates.     

Kinetics of the inhibition of free and elastin-bound human pancreatic elastase by alpha 1-proteinase inhibitor and alpha 2-macroglobulin

At pH 8.0 and 25 degrees C alpha 1-proteinase inhibitor and alpha 2-macroglobulin bind human pancreatic elastase with rate constants of 4.7.10(5) M-1.s-1 and 6.4.10(6) M-1.s-1, respectively. The corresponding delay times of elastase inhibition in plasma are 0.4 s and 0.2 s, respectively, indicating that both inhibitors may act as physiological antielastases. Elastin impairs the elastase inhibitory capacity of alpha 1-proteinase inhibitor and alpha 2-macroglobulin. In presence of human elastin, the former behaves like a slow-binding elastase inhibitor, with a rate constant of about 260 M-1.s-1. In contrast, alpha 2-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin.     

Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach

A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.     

The Effect of Static Stretch on Elastin Degradation in Arteries

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries [1]. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11±0.04 h⁻¹) is almost twice that of digestion without stretch (0.069±0.028 h⁻¹). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation.     

Influence of elastin on the inhibition of leucocyte elastase by alpha 1-proteinase inhibitor and bronchial inhibitor. Potent inhibition of elastin-bound elastase by bronchial inhibitor.

We have investigated the effect of human lung elastin on the inhibition of human leucocyte elastase by human alpha 1-proteinase inhibitor and bronchial inhibitor. Elastin was unable to dissociate the elastase-inhibitor complexes during the 150 min of the elastolysis reaction. When elastase was added to mixtures of elastin and alpha 1-proteinase inhibitor, it was fully bound to the latter. The competition between elastin and bronchial inhibitor was also in favour of the latter, but a 1.5 molar excess of inhibitor over elastase was required to achieve total binding of the enzyme. About 25% of elastin-bound elastase was found to be resistant to the inhibitory effect of alpha 1-proteinase inhibitor. The major isoenzyme and the mixture of the three minor isoenzymes of elastase exhibited similar behaviour. By contrast, bronchial inhibitor was as efficient in inhibiting the elastin-bound elastase as it was in inhibiting the free enzyme. This inhibitor was also able to inhibit fully the fraction of elastin-bound elastase that was resistant to alpha 1-proteinase inhibitor. We also describe a rapid procedure for the isolation of gram quantities of alpha 1-proteinase inhibitor.     

Effect of pancreatic and leukocyte elastase on hydraulic conductivity in lung interstitial segments.

Elastase-induced changes in flow were used to quantify the degradation of lung interstitial elastin. Degassed rabbit lungs were inflated with silicon rubber via airways and vessels. The lungs were cut into 1-cm-thick sections. Two chambers were bonded to each section to enclose the interstitium surrounding an arterial segment. Flow of albumin solution (0-5 g/dl) between the chambers was followed by that of the albumin solution with 0.25 g/dl pancreatic elastase solution. Driving pressure was 5 cmH(2)0, and mean interstitial pressure was either 0 or 10 cmH(2)O. Elastase caused an increase in flow in approximately 70% of the interstitial segments and a reduction in flow in the remaining segments. The elastase-induced response in flow was independent of both albumin concentration and mean interstitial pressure. Leukocyte elastase (5 units/dl) produced flow responses similar to those of 0.25 g/dl pancreatic elastase. The increased flow of leukocyte elastase was reduced by a subsequent flow with 0.25 g/dl pancreatic elastase but enhanced by a subsequent flow with a 10-fold lower concentration. A change in the order of the elastase flows reversed the concentration-dependent responses. This behavior suggests a complex interaction among the interstitial fibers after degradation by pancreatic and leukocyte elastase. Endogenous elastase-induced increases in interstitial permeability might affect blood-lymph barrier permeability, whereas elastase-induced cessation of flow might be related to the alveolar septal wall destruction observed in emphysema.     

Effect of neutrophil cathepsin G on elastin degradation by neutrophil elastase

Human neutrophil cathepsin G was found to be unable to significantly stimulate the degradation of either bovine or human elastin by neutrophil elastase, using four different procedures to monitor digestion. A range of stimulations from 1.1 to 2.9-fold was found, with a 2.0-fold stimulation being the average found with the assays tested. These results contrast with those reported by Boudier et al. [(1981) J. Biol. Chem. 256, 10256-10258] who reported a five- to seven-fold stimulation of elastolysis of human lung elastin by cathepsin G, when present at a 2:1 molar ratio relative to elastase. Significantly, we found little stimulation of elastolysis with either human or bovine lung elastin as substrate while Boudier et al. found stimulation only with the human elastin. Thus, it would appear that cathepsin G does not play a predominant role as an elastolytic enzyme; rather, its role in this case may be one of binding to non-productive sites on the elastin surface.     

The role of elastin in the mechanical properties of skin

The elastin fibers of rat skin samples were degraded by the use of a purified preparation of elastase to which soybean inhibitor was added, preventing the collagenolytic activity of the elastase on collagen. Control experiments ascertained degradation of elastin and no effect on collagen. The mechanical properties of the skin samples were studied before and after the enzymatic treatment and differences ascribed to the degraded elastin fibers. Elastin plays a role in the mechanical behaviour of rat skin at small stress values and small deformations. Especially, the elastin fibers are responsible for the recoiling mechanism after a stress or deformation has been applied.     

The effect of elastin damage on the mechanics of the aortic valve

Porcine bioprosthetic heart valves degenerate and fail mechanically through a mechanism that is currently not well understood. It has been suggested that damage to the elastin component of prosthetic valve cusps could be responsible for changes in the mechanical function of the valve that would predispose it to increased damage and ultimate failure. To determine whether damage to elastin can produce the structural and mechanical changes that could initiate the process of bioprosthetic valve degeneration, we developed an elastase treatment protocol that fragments elastin and negates its mechanical contribution to the valve tissue. Valve cusps were mechanically tested before and after digestion to measure the mechanical changes resulting from elastin damage. Elastin damage produced a decrease in radial and circumferential extensibility (from 43 to 18% strain radially and 12 to 7% strain circumferentially), with a slight increase in stiffness (1.3-2.6kN/m for radial and 10.6-11.9kN/m for circumferential directions). Digestions with trypsin, which does not cleave elastin, confirmed that the changes in mechanics of the circumferential samples were likely due to the nonspecific removal of proteoglycans by elastase, while the changes in the radial samples were indeed due to elastin damage. Removing the mechanical contribution of elastin alters the mechanical behavior of the aortic valve cusp, primarily in the radial direction. This finding implies that damage to elastin will distend the cusps, reduce their extensibility, and increase their stiffness. Damage to elastin may therefore contribute to the degeneration and failure of prosthetic valves.