UVA light stimulates the production of cathepsin G and elastase-like enzymes by dermal fibroblasts: a possible contribution to the remodeling of elastotic areas in sun-damaged skin

Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sun-exposed and non-exposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast-like cells from specimens of sun-exposed areas. Little or no reaction was found in biopsies of sun-protected skin. Fibroblast cultures from sun-exposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sun-protected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sun-protected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastase-like activity expressed by fibroblasts can be attributed to at least two enzymes.     

Mechanical and failure properties of extracellular matrix sheets as a function of structural protein composition

The goal of this study was to determine how alterations in protein composition of the extracellular matrix (ECM) affect its functional properties. To achieve this, we investigated the changes in the mechanical and failure properties of ECM sheets generated by neonatal rat aortic smooth muscle cells engineered to contain varying amounts of collagen and elastin. Samples underwent static and dynamic mechanical measurements before, during, and after 30 min of elastase digestion followed by a failure test. Microscopic imaging was used to measure thickness at two strain levels to estimate the true stress and moduli in the ECM sheets. We found that adding collagen to the ECM increased the stiffness. However, further increasing collagen content altered matrix organization with a subsequent decrease in the failure strain. We also introduced collagen-related percolation in a nonlinear elastic network model to interpret these results. Additionally, linear elastic moduli correlated with failure stress which may allow the in vivo estimation of the stress tolerance of ECM. We conclude that, in engineered replacement tissues, there is a tradeoff between improved mechanical properties and decreased extensibility, which can impact their effectiveness and how well they match the mechanical properties of native tissue.     

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.     

Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.     

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.     

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.     

Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase. An in vitro model of elastolytic injury

Summary A neonatal rat aorta smooth muscle cell culture system with a unique elastin-rich extracellular matrix was used as a model substrate for elastases. To study the susceptibility to solubilization of insoluble elastin, cultures were incubated in the presence of human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE) and in the absence of serum for periods up to 45 min. Both the incubation media and cell layers were then assessed for elastin and collagen markers, total protein, and lactate dehydrogenase (LDH). Although HNE and PPE exhibited comparable activity against elastin purified from the cell layer, HNE exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity using intact cell layers as compared with the purified elastin, whereas PPE exhibited only a 1.5- to 2.5-fold reduction. This effect could not satisfactorily be explained as preferential inhibition of HNE activity in the culture system, because the amount of protein solubilized by HNE was 59% that of PPE. The mean elastin content of PPE-solubilized protein was 110% that of the elastin content of the corresponding cell layer; the value for HNE-solubilized protein was only 16%. Thus, the amount of elastin per microgram of solubilized protein for HNE was 15% that for PPE. Possible explanations for the greatly diminished elastolytic activity of HNE in the culture system include the preference of HNE for other substrates in the cell layer, the inability of HNE to penetrate sufficiently into the cell layer, and the presence of sulfated glycosaminoglycans in the vicinity of the elastin that act in an inhibitory fashion. Although there was extensive proteolytic damage to the extracellular matrix, LDH and DNA measurements indicated that little loss of cells or cell viability occurred. The observed differences in elastolytic activity of HNE and PPE in the culture system parallel the relative emphysema-inducing potency of the elastases in the hamster model of elastase-induced emphysema. Supported by National Heart, Lung and Blood Institute, Bethesda, MD, grants NIH-HL-25229, HL-19717, and HL-33522. Presented in part at the April 1985 meeting of the Federation of American Societies for Experimental Biology.     

Impact of microvascular circulation on peripheral lung stability

The involvement of pulmonary circulation in the mechanical properties was studied in isolated rat lungs. Pulmonary input impedance (ZL) was measured at a mean transpulmonary pressure (Ptpmean) of 2 cmH2O before and after physiological perfusion with either blood or albumin. In these lungs and in a group of unperfused lungs, ZL was also measured at Ptpmean values between 1 and 8 cmH2O. Airway resistance (Raw) and parenchymal damping (G) and elastance (H) were estimated from ZL. End-expiratory lung volume (EELV) was measured by immersion before and after blood perfusion. The orientation of the elastin fibers relative to the basal membrane was assessed in additional unperfused and blood-perfused lungs. Pressurization of the pulmonary capillaries significantly decreased H by 31.5 +/- 3.7% and 18.7 +/- 2.7% for blood and albumin, respectively. Perfusion had no effect on Raw but markedly altered the Ptpmean dependences of G and H < 4 cmH2O, with significantly lower values than in the unperfused lungs. At a Ptpmean of 2 cmH2O, EELV increased by 31 +/- 11% (P = 0.01) following pressurization of the capillaries, and the elastin fibers became more parallel to the basal membrane. Because the organization of elastin fibers results in smaller H values of the individual alveolus, the higher H in the unperfused lungs is probably due to a partial alveolar collapse leading to a loss in lung volume. We conclude that the physiological pressure in the pulmonary capillaries is an important mechanical factor in the maintenance of the stability of the alveolar architecture.     

Heparan sulfate depletion within pulmonary fibroblasts: implications for elastogenesis and repair

We investigated the role of sulfated proteoglycans in regulating extracellular matrix (ECM) deposition in pulmonary fibroblast cultures. Fibroblast cultures were subject to pharmacologic and enzymatic interventions to modify sulfated proteoglycan levels. Native and proteoglycan-depleted fibroblasts were treated with porcine pancreatic elastase at 2-4-day intervals and the elastase-mediated release of fibroblast growth factor 2 (FGF-2) and glycosaminoglycans was determined. Elastase treatment released significantly less FGF-2 and glycosaminoglycans (GAG) from PG-depleted fibroblasts with respect to native cells. Equilibrium ligand binding studies indicated that 125I FGF-2 binding at both cell surface receptor and heparan sulfate proteoglycan sites was reduced to different extents based on the method of proteoglycan depletion. Quantitation of elastin protein and message levels indicated that biological sulfation is required for the proper incorporation of tropoelastin into the extracellular matrix. These results suggest that sulfated proteoglycans play a central role in modulating pulmonary fibroblast extracellular matrix composition and are important mediators of elastolytic injury.     

Co-operation between plasmin and elastase in elastin degradation by intact murine macrophages.

Intact, thioglycollate-stimulated murine macrophages cultured on an insoluble [3H]-elastin substratum progressively hydrolysed the elastin. Cell lysates had little activity. We compared the effect of various proteinase inhibitors on elastinolysis by either live cells or cell-free, elastase-rich conditioned medium. Only known inhibitors of macrophage elastase blocked the activity of elastase-rich cell-conditioned medium whereas inhibitors of cathepsin B also suppressed intact cell activity. Serum proteinase inhibitors blocked cell-derived soluble elastase activity but not intact cell elastolytic activity. We also observed that plasminogen added to the cell cultures markedly increased elastinolysis by live macrophages or cell-free elastase-rich medium. Purified plasmin alone had no measurable effect on native elastin. Additional experiments indicated that the plasmin enhancement was due to elastin-dependent activation of latent macrophage elastase. These results indicate that live macrophage elastinolysis is a co-operative process involving multiple proteinases, especially a cysteine proteinase(s) and elastase. Plasmin may be a physiological activator of latent macrophage elastase.     

Induction of the myofibroblast phenotype following elastolytic injury to mouse lung

The repair of alveolar structures following endotracheal administration of porcine pancreatic elastase (PPE) to mice involves the coordinated deposition of new matrix elements. We determined the induction of the myofibroblast phenotype following elastolytic injury to mouse lung by examining the expression of α-smooth muscle actin (α-SMA) by immunohistochemistry. We also examined elastin and α1(I) collagen mRNA expression by in situ hybridization. Changes in airspace dimensions were assessed by determining mean linear intercept. In untreated mice, α-SMA was localized to vascular structures and large airways, with no detectable expression in alveolar units. PPE induced α-SMA expression in damaged areas surrounding large vessels, in septal remnants, and in the opening ring of alveolar ducts. Elastin and α1(I) collagen mRNA expression were up-regulated in residual alveolar structures and septal walls. PPE dose-response studies indicated that α1(I) collagen and elastin mRNA expression were not induced in areas of normal lung adjacent to damaged lung. The administration of low dose PPE resulted in increased α-SMA protein and elastin mRNA expression in the cells comprising the opening ring of alveolar ducts. Our data suggest that repair mechanisms following elastolytic injury are confined to overtly damaged alveolar structures and involve the induction of the myofibroblast phenotype.     

The viscoelastic basis for the tensile strength of elastin

Purified aortic elastin displays failure behaviour characteristic of an amorphous, noncrystalizing elastomer with failure properties showing a strong dependence on viscoelastic behaviour. Tensile breaking stresses and breaking strains measured over a range of temperatures, hydration levels, and strain rates are reducible to single curves by the application of shift factors obtained from dynamic mechanical tests. The breaking stress of rubbery elastin is similar to that found in other elastomers, but glassy elastin is about an order of magnitude less strong than expected. We suggest elastin's ability to be strengthened through viscous dissipation of strain energy and crack tip blunting is limited by its fibrillar structure.     

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.     

Dynamic mechanical properties of elastin.

The dynamic mechanical properties of water-swollen elastin under physiological conditions have been investigated. When elastin is tested as a colsed, fixed-volume system, mechanical data could be temperature shifted to produce master curves. Master curves for elastin hydrated at 36°C (water content, 0.46 g water/g protein) and 55°C (water content, 0.41 g/g) were constructed, and in both cases elastin goes through a glass transition, with the glass transition temperatures of -46 and -21°C, respectively. Temperature shift data used to construct the master curves follow the WLF equation, and the glass transition appears to be characteristic of an amorphous, random-polymer network. For elastin tested as an open, variable-volume system free to change its swollen volume as temperature is changed, dynamic mechanical properties appear to be virtually independent of temperature. No glass transition is observed because elastin swelling increases with decreased temperature, and the increase in water content shifts elastin away from its glass transition. It is suggested that the hydrophobic character of elastin, which gives rise to the unusual swelling properties of elastin, evolved to provide a temperature-independent elastomer for the cold-blooded, lower vertebrates.     

Differential effects of static and cyclic stretching during elastase digestion on the mechanical properties of extracellular matrices.

Enzyme activity plays an essential role in many physiological processes and diseases such as pulmonary emphysema. While the lung is constantly exposed to cyclic stretching, the effects of stretch on the mechanical properties of the extracellular matrix (ECM) during digestion have not been determined. We measured the mechanical and failure properties of elastin-rich ECM sheets loaded with static or cyclic uniaxial stretch (40% peak strain) during elastase digestion. Quasistatic stress-strain measurements were taken during 30 min of digestion. The incremental stiffness of the sheets decreased exponentially with time during digestion. However, digestion in the presence of static stretch resulted in an accelerated stiffness decrease, with a time constant that was nearly 3 x smaller (7.1 min) than during digestion alone (18.4 min). These results were supported by simulations that used a nonlinear spring network model. The reduction in stiffness was larger during static than cyclic stretch, and the latter also depended on the frequency. Stretching at 20 cycles/min decreased stiffness less than stretching at 5 cycles/min, suggesting a rate-dependent coupling between mechanical forces and enzyme activity. Furthermore, pure digestion reduced the failure stress of the sheets from 88 +/- 21 kPa in control to 29 +/- 15 kPa (P < 0.05), while static and cyclic stretch resulted in a failure stress of 7 +/- 5 kPa (P < 0.05). We conclude that not only the presence but the dynamic nature of mechanical forces have a significant impact on enzyme activity, hence the deterioration of the functional properties of the ECM during exposure to enzymes.     

Viscoelastic and dynamic nonlinear properties of airway smooth muscle tissue: roles of mechanical force and the cytoskeleton

The viscoelastic and dynamic nonlinear properties of guinea pig tracheal smooth muscle tissues were investigated by measuring the storage (G') and loss (G") moduli using pseudorandom small-amplitude length oscillations between 0.12 and 3.5 Hz superimposed on static strains of either 10 or 20% of initial length. The G" and G' spectra were interpreted using a linear viscoelastic model incorporating damping (G) and stiffness (H), respectively. Both G and H were elevated following an increase in strain from 10 to 20%. There was no change in harmonic distortion (K(d)), an index of dynamic nonlinearity, between 10 and 20% strains. Application of methacholine at 10% strain significantly increased G and H while it decreased K(d). Cytochalasin D, isoproterenol, and HA-1077, a Rho-kinase inhibitor, significantly decreased both G and H but increased K(d). Following cytochalasin D, G, H, and K(d) were all elevated when mean strain increased from 10 to 20%. There were no changes in hysteresivity, G/H, under any condition. We conclude that not all aspects of the viscoelastic properties of tracheal smooth muscle strips are similar to those previously observed in cultured cells. We attribute these differences to the contribution of the extracellular matrix. Additionally, using a network model, we show that the dynamic nonlinear behavior, which has not been observed in cell culture, is associated with the state of the contractile stress and may derive from active polymerization within the cytoskeleton.     

Age-related changes in the content of fibrous proteins in the rat tissues.

An attempt was made to establish a relationship between the content of elastin and collagen in the rat tissues during the process of aging. The content of collagen fractions and elastin in the rat liver, lung and skin, as well as the elastolytic activity of blood serum were determined. It was found that the concentration of elastin as well as the elastolytic activity of blood serum are increasing during the maturation of rats and the total collagen content is increasing too. After the animals reached the age from twelve to twenty four months--above mentioned values began to decrease. It is concluded that the changes in the content of the two fibrous proteins of the connective tissue depend on age.     

Hypoxia suppresses elastin repair by rat lung fibroblasts

Macrophage and neutrophil proteinases damage lung elastin, disrupting alveolar epithelium and filling alveoli with inflammatory exudate. Alveolar collapse and regional hypoxia occur. Whether low oxygen tension alters fibroblast-mediated lung repair is unknown. To determine the effect of chronic hypoxia on repair of enzyme-induced elastin disruption, primary rat lung fibroblasts produced elastin matrix for 5 wk before treatment with porcine pancreatic elastase (PPE). After exposure to PPE or saline, cultures recovered for 2 wk in normoxia (21% O(2)) or hypoxia (3% O(2)). Hypoxia suppressed regeneration of hot alkali-resistant elastin, achieving only 49% of the repair achieved in normoxic cultures. Vascular smooth muscle cells and lung fibroblasts repair elastin by two pathways: de novo synthesis and salvage repair. Although both pathways were affected, hypoxia predominantly inhibited de novo synthesis, decreasing formation of new elastin matrix by 63% while inhibiting salvage repair by only 36%. Prolonged hypoxia alone downregulated steady-state levels of elastin mRNA by 45%, whereas PPE had no significant effect on elastin gene expression. Electron microscopy documented preservation of intracellular organelles and intact nuclei. Together, these data suggest that regional hypoxia limits lung elastin repair following protease injury at least in part by inhibiting elastin gene expression.     

Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity.

A 22-kilodalton protein purified from the culture supernatant fraction of Pseudomonas aeruginosa (strains PA220 and PAO1) was found to enhance the elastolytic activity of purified P. aeruginosa elastase. N-terminal sequence analysis identified the protein as a fragment of the lasA gene product (P.A. Schad and B.H. Iglewski, J. Bacteriol. 170:2784-2789, 1988). However, comparative analysis with the reported LasA sequence indicated that the purified LasA fragment is longer than the deduced sequence reported. The purified LasA fragment had minimal elastolytic and proteolytic activity and did not enhance the proteolytic activity of purified elastase, yet enhanced the elastolytic activity more than 25-fold. The LasA fragment was found to also enhance the elastolytic activities of thermolysin, human neutrophil elastase, and proteinase K. The results presented here suggest that the LasA protein interacts with the elastin substrate rather than modifying elastase.