Elastin metabolism during recovery from impaired crosslink formation

Accelerated proteolysis of tropoelastin and elastin occurs in the arteries of chicks rendered nutritionally copper-deficient. The process results in part from decreased elastin crosslinking. Repletion of copper-deficient chicks with copper causes a deposition of elastin that is proteinase resistant. Resistance to proteolysis is conferred within 48 h of dietary copper repletion. Deposition of aorta elastin to near normal values occurs after 3-4 days in copper-repleted chicks. Moreover, elastolysis was enhanced when the content of dehydrolysinonorleucine in elastin was abnormally low. The chemical modification of lysyl residue in elastin by citroconylation, however, did not influence the rate of elastolysis. We have shown previously that tropoelastin messenger RNA activity and synthesis are not influenced by dietary copper deprivation (1986, Biochem. J. 236, 17-23). Rather, as demonstrated herein, the decrease in elastin content in arteries of copper-deficient birds appears to be more the result of enhanced degradation. Restoration of normal crosslinking restores deposition and imparts resistance to elastolysis. Moreover, serum appears to be a good source of elastolytic proteinases when the elastin substrate is partially or abnormally crosslinked.     

Susceptibility of baboon aorta elastin to proteolysis

Elastin was purified from baboon aorta using Achromobacter collagenase and its susceptibility to proteolysis by various enzymes was studied. Human leukocyte elastase (HLE) hydrolysed baboon aortic elastin 8 times faster than human cathepsin G. Bovine chymotrypsin had virtually no activity against this substrate. The kinetic constants V and [S50] of aortic elastin hydrolysis by HLE (0.15 microM) were 0.00286 mg x ml-1 x min-1 and 0.158 mg x ml-1, respectively. One mg of this elastin could be saturated with 5.6 micrograms of HLE. As with elastins isolated from other sources, the hydrolysis of baboon aortic elastin by HLE was highly sensitive to ionic strength, and a biphasic effect was obtained with increasing NaCl concentrations. A nearly 2-fold stimulation of elastolysis was observed at a 0.15M NaCl concentration. Further increase in ionic strength led to a continuous decrease of the rate of elastolysis which paralleled the decrease of adsorption of elastase to baboon aortic elastin. Cathepsin G, but not bovine alpha-chymotrypsin, was able to stimulate the rate of hydrolysis of baboon aortic elastin by HLE. A 1.7 fold stimulation was observed for a 1:1 molar ratio of the two proteinases and rose to 2.1 for a HLE/Cat. G ratio equal to 8.     

Alterations in the chain dynamics of insoluble elastin upon proteolysis by serine elastases

The high temperature dielectric relaxations of purified and elastolized ligamentum nuchae elastin in the dry state have been investigated by thermally stimulated depolarization current spectrometry, with an equivalent frequency comprised between 10(-2) and 10(-3) Hz. A main relaxation mode, located close to 150 degrees C and attributed to the dielectric manifestation of a glass transition, is found for all samples. After decomposition by the fractional polarization method, the analysis of the high temperature mode shows the existence of two relaxation mechanisms: a cooperative one, associated with flexible zones of the protein, and an isoenthalpic one, corresponding to more ordered and constrained zones. The activation parameters of the two mechanisms are dependent on the extent of elastolysis and on the nature of enzyme (pancreatic elastase vs leukocyte elastase). Both enzymes influence the dielectric behavior of elastin in a similar way: the activation enthalpy maximum of the relaxing units located in the flexible zones, characteristic of the cooperative length, decreases with increasing hydrolysis. Moreover, the isoenthalpic mechanism becomes cooperative at the highest extent of elastolysis, which highlights release of constraints in ordered zones. Nevertheless, the differences found between the two enzymatic hydrolyses are characteristic of distinct sites of cleavage in the elastin network.     

Elastolysis of kappa-elastin sub-fractions by pancreatic and leukocyte elastases

Kappa-elastin peptides, obtained from insoluble elastin by organo-alkaline hydrolysis, were fractioned by gel filtration on Biogel agarose. Rates of hydrolysis by pancreatic and leukocyte elastases of the fractions were measured using a conductometric method. Kinetics obey to Michaelis-Menten model for both substrates and enzymes. KM and Vmax values derived from Lineweaver-Burk plots indicate that, if KM remains quite constant, differences were observed in catalytic rates. The kcat values decreased with molecular-weight, the high-molecular-weight kappa-elastin peptides being hydrolyzed 3 to 5 times faster than the low-molecular-weight ones. Apparent differences between potentiometric (pH-Stat) and conductometric results were discussed, in relation with buffer capacity of soluble and insoluble elastins.     

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.     

Tropoelastin production and tropoelastin messenger RNA activity. Relationship to copper and elastin cross-linking in chick aorta.

The elastin content of the chick thoracic aorta increases 2--3-fold during the first 3 weeks post-hatching. The deposition of elastin requires the covalent cross-linking of tropoelastin by means of lysine-derived cross-links. This process is sensitive to dietary copper intake, since copper serves as cofactor for lysyl oxidase, the enzyme that catalyses the oxidative deamination of the lysine residues involved in cross-link formation. Disruption of cross-linking alters tissue concentrations of both elastin and tropoelastin and results in a net decrease in aortic elastin content. Autoregulation of tropoelastin synthesis by changes in the pool sizes of elastin or tropoelastin has been suggested as a possible mechanism for the diminished aortic elastin content. Consequently, dietary copper deficiency was induced to study the effect of impaired elastin cross-link formation on tropoelastin synthesis. Elastin in aortae from copper-deficient chicks was only two-thirds to one-half the amount measured in copper-supplemented chicks, whereas copper-deficient concentrations of tropoelastin in aorta were at least 5-fold higher than normal. In spite of these changes, however, increased amounts of tropoelastin, copper deficiency and decreased amounts of elastin did not influence the amounts of functional elastin mRNA in aorta. Likewise, the production of tropoelastin in aorta explants was the same whether the explants were taken from copper-sufficient or -deficient birds. The lower accumulation of elastin in aorta from copper-deficient chicks appeared to be due to extracellular proteolysis, rather than to a decrease in the rate of synthesis. Electrophoresis of aorta extracts, followed by immunological detection of tropoelastin-derived products, indicated degradation products in aortae from copper-deficient birds. In extracts of aortae from copper-sufficient chicks, tropoelastin was not degraded and appeared to be incorporated into elastin without further proteolytic processing.     

Use of enzymolysis techniques in studying the mechanical properties of connective tissue components.

The optimum conditions for the selective removal of elastin from connective tissues are described. The process, elastolysis, consists of incubating small samples of connective tissue in buffered saline at ph=8.6 containing 300 microgram/me of a 50-50% mixture of elastase with trypsin inhibitor, for 5-6 hours at room temperature. This process, complimented with other processes for selective removal of lipids, or mucopolysaccharides, or collagen, enables one to examine the contribution of the various components of the connective tissue to its mechanical function. The elastolysis was tested with aortic, valvular and tendon tissues from human, bovine and canine species and it was found that in tensile stress experiments, collagen was unaffected while the low-stress contribution of elastin disappeared.     

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).     

Silicon facilitation of copper utilization in the rat

An eight-week, 2 x 4 factorial rat experiment using two levels of dietary copper and four levels of dietary silicon was conducted to further delineate a previously observed silicon-copper interaction in which silicon appears to mimic copper in its effect on the composition of the aorta. Dietary copper concentrations were 1.4 (deficient) and 5.4 (adequate) mg/kg diet, and silicon concentrations were 5, 135, 270, and 540 mg/kg diet. Compared with the lowest level of silicon and copper, weight gains were 15.5% higher for rats fed 540 mg silicon/kg diet and 14.3% higher for those fed 5.4 mg copper/kg diet. The growth-promoting effects of silicon and copper were additive. Evidence that silicon elevated the copper status of copper-deficient rats includes an increase in packed-cell volume by 540 mg silicon/kg diet in the otherwise packed-cell volume-depressed, copper-deficient rats, accompanied by a trend toward higher hemoglobin values and lower relative heart weights. In the copper-adequate rats, evidence that 540 mg silicon/kg diet elevated their copper status includes a two-fold increase in the blood-plasma copper concentration, a three-fold increase in ceruloplasmin activity, and an increase in cardiac, renal, and hepatic copper concentrations. In addition, 540 mg silicon/kg diet resulted in higher aortic dry mass and aortic elastin content in both copper-deficient and copper-adequate rats. While dietary silicon concentrations of 135, 270, and 540 mg/kg diet were all effective in increasing aortic elastin in the copper-adequate rats, only 540 mg silicon/kg diet increased aortic elastin in the copper-deficient rats. These data indicate that some of the metabolic effects attributed to silicon may be manifested through a silicon-facilitated increase in copper utilization.     

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.     

Stimulation of human granulocyte elastase by platelet factor 4 and heparin

Highly purified platelet factor 4 (PF₄) was found to be a potent stimulator of human granulocyte elastase activity against native elastin and solubilized α elastin. Heparin neutralized this stimulation of elastolysis by PF₄, but independently stimulated granulocyte elastase activity. Chondroitin sulfate, a constituent of the PF₄ carrier molecule, also stimulated granulocyte elastase activity. The stimulation of granulocyte elastase by PF₄ occurs at known serum concentrations of PF₄.     

Solubilized elastin substrate for continuous fluorimetric assay of kinetics of elastases

Elastolysis is central to progression of emphysema and aortic aneurysms. Characterization of steady-state enzyme kinetics of elastolysis is fettered by the insolubility of mature elastin and the polydispersity of solubilized elastin. We prepared a fluor-tagged, 100-kDa fraction (fEln-100) from commercial α-elastin. It is soluble, less heterogeneous in mass, cross-linked like mature elastin, and likely to retain the capacity of α-elastin to self-assemble. fEln-100 has introduced the ability to compare quantitatively the apparentk_cat and K_m of elastases. For example, metalloelastase (MMP-12) displays higher apparent affinity for fEln-100, while MMP-2 displays faster catalytic turnover.     

Molecular mobility of elastin: effect of molecular architecture

The thermal and dielectric properties of elastin and two soluble derivatives (kappa-elastin and derived elastin peptides from enzymatic elastolysis) were investigated in the freeze-dried state in a wide temperature range (from -180 to +220 degrees C). The glass transition of these amorphous proteins was studied by differential scanning calorimetry (DSC). The dielectric relaxations of both proteins were followed by thermally stimulated currents (TSC), an isochronal dielectric spectrometry running at variable temperature, analogous to a low-frequency spectroscopy (10(-3)-10(-2) Hz) and by dynamic dielectric spectroscopy (DDS), performed isothermally with the frequency varying from 10(-2) to 3 x 10(6) Hz. The combination of TSC and DDS experiments and the determination of the activation parameters of the relaxation times inform about the molecular mobility of the proteins, both in the glassy state and in the liquid state. Major differences between the relaxation behavior of elastin and its soluble derivatives have been discussed and correlated with the molecular architecture of the proteins.     

Skin atrophy in cytoplasmic SOD-deficient mice and its complete recovery using a vitamin C derivative

Intrinsic skin ageing is characterized by atrophy and loss of elasticity. Although the skin hypertrophy induced by photoageing has been studied, the molecular mechanisms of skin atrophy during ageing remain unclear. Here, we report that copper/zinc superoxide dismutase (CuZn-SOD)-deficient mice show atrophic morphology in their skin. This atrophy is accompanied by the degeneration of collagen and elastic fibers, and skin hydroxyproline is also significantly reduced in deficient mice. These imply that the dysfunction of collagen and elastin biosynthesis are involved in the progression of skin thinning. Furthermore, transdermal administration of a vitamin C derivative which can permeate through the membrane, completely reversed the skin thinning and deterioration of collagen and elastin in the mutant mice. These indicate that the vitamin C derivative is a powerful agent for alleviating skin ageing through regeneration of collagen and elastin. The CuZn-SOD-deficient mice might be applicable to evaluation of therapeutic medicines against skin ageing.     

Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy

Objectives

Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis.

Methods

We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM).

Results

IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated Marfan aorta: Ad.hTIMP-1 p = 0.902; control Ad.β-Gal. p = 0.165). The virus-untreated and not transplanted mgR/mgR aorta revealed a significant increase of albumin diffusion through the endothelial barrier (p = 0.037). TEM analysis of adenovirus-exposed mgR/mgR aortas displayed disruption of the basement membrane and basolateral space.

Conclusions

Murine Marfan aortic grafts developed severe inflammation after adenoviral contact. We demonstrated that fibrillin-1 deficiency is associated with relevant dysfunction of the endothelial barrier that enables adenovirus to induce vessel-harming inflammation. Endothelial dysfunction may play a pivotal role in the development of the vascular phenotype of Marfan syndrome.     

The effect of agarose and dexamethasone on the nature and production of extracellular matrix components by elastic cartilage chondrocytes

Chondrocytes isolated enzymatically from rabbit ear cartilage, were cultivated in vitro in the presence of 2% agarose or 0.1 mumol/l dexamethasone. Freshly-isolated chondrocytes suspended in either Eagle's medium or 2% agarose were auto-transplanted intramuscularly. Samples were then examined by light microscopy and transmission electron microscopy. The cells cultivated in vitro rapidly formed confluent multiple overlapping layers filled with a loose matrix consisting of single collagen fibres, proteoglycans and scarce elastic fibres. The number and maturity of the elastic fibres increased substantially after dexamethasone was added. The chondrocytes in intramuscular transplants produced a larger amount of intercellular matrix with many elastic fibres than those cultured in vitro. Addition of agarose to in vitro and in vivo systems selectively suppressed the elastin production but did not diminish the production of elastic fibre microfibrils and other matrix components. This made cultures and transplants of elastic chondrocytes resemble rather hyaline cartilage than the original tissue. It seems that the lack of elastin in the matrix does not result simply from inhibition of elastin secretion or increased elastolysis. It may be related to a reversible change of genetic expression of elastic cartilage chondrocytes under the influence of agarose.     

The Receptor for Advanced Glycation End Products (RAGE) Contributes to the Progression of Emphysema in Mice

Several recent clinical studies have implied a role for the receptor for advanced glycation end products (RAGE) and its variants in chronic obstructive pulmonary disease (COPD). In this study we have defined a role for RAGE in the pathogenesis of emphysema in mice. RAGE deficient mice (RAGE-/-) exposed to chronic cigarette smoke were significantly protected from smoke induced emphysema as determined by airspace enlargement and had no significant reduction in lung tissue elastance when compared to their air exposed controls contrary to their wild type littermates. The progression of emphysema has been largely attributed to an increased inflammatory cell-mediated elastolysis. Acute cigarette smoke exposure in RAGE-/- mice revealed an impaired early recruitment of neutrophils, approximately a 6-fold decrease compared to wild type mice. Hence, impaired neutrophil recruitment with continued cigarette smoke exposure reduces elastolysis and consequent emphysema.     

Further studies on Pseudomonas aeruginosa LasA: analysis of specificity

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave beta-casein. Preliminary analysis of beta-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.     

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.     

Alveolar macrophage-mediated elastolysis: roles of matrix metalloproteinases,cysteine, and serine proteases

Chronic obstructive pulmonary disease (COPD) is a common lung disease with cigarette smoking as the major etiological factor, but only 15% of smokers develop COPD. Destruction of lung elastin observed in COPD is mediated by many enzymes, including cysteine, serine, and matrix metalloproteinases (MMP). The contribution of these enzymes to the lung elastolytic load, released from alveolar macrophages collected from nonsmokers, healthy smokers, and COPD patients, was examined by radiolabeled elastin as substrate in the presence of specific enzyme inhibitors. The activity of MMP was further examined by zymography and Western blotting. COPD macrophages degraded more elastin than either of the other groups. Elastolysis was greatest in the initial 24 h. Through the 72-h culture period, the contribution to elastolysis of serine elastases decreased, MMP increased, and cysteine elastases remained constant. The increased release of elastolytic enzymes in COPD subjects may explain why some smokers develop COPD. This difference may be due to unknown susceptibility factors. Serine proteases play a significant role; however, other enzymes, particularly the MMP, deserve further investigation.