Demonstration of a precursor-product relationship between soluble and cross-linked elastin, and the biosynthesis of the desmosines in vitro.

Direct evidence showing that a soluble form of elastin is the precursor of cross-linked elastin was obtained from pulse-chase experiments using chick embryo aortas and by demonstrating the conversion of soluble elastin into cross-linked elastin in a cell-free system. Acetic acid extracts of embryonic chick aorta pulse-labeled with [14C]lysine contain two radioactive proteins of molecular weights 74,000 and 138,000 which have been identified previously as soluble elastin and the pro-alpha chain of collagen, respectively. In pulse-chase experiments, the radioactivity incorporated in the soluble elastin during the pulse with [14C]lysine disappeared during a 24-hour chase with [12C]lysine and 89% of that which disappeared was accounted for in the desmosines of alkali-insoluble elastin. The disappearance of the radioactivity from the soluble fraction and its appearance in the desmosines of elastin were inhibited by beta-aminopropionitrile, a specific inhibitor of the cross-linking enzyme lysyl oxidase. In addition in vitro experiments, it was shown that the radioactivity in the desmosines of elastin can arise from that present in an acid-soluble precursor protein. This precursor protein is soluble elastin, as demonstrated by the formation of desmosines when a homogeneous preparation of soluble elastin was incubated with purified lysyl oxidase.     

Recombinant interleukin-1 beta inhibits elastin formation by a neonatal rat lung fibroblast subtype

The effect of recombinant interleukin-1 beta (rIL-1 beta) on elastin accumulation by lipid-laden interstitial cells (LIC) derived from neonatal rat lung was examined. The LIC, a fibroblast subtype, synthesized large amounts of elastin which was deposited into the extracellular matrix. This elastin was alkali-resistant and had an amino acid composition typical of adult rat elastin. Treatment of lipid-laden interstitial cell cultures with rIL-1 beta at 100 pg/ml caused a dramatic decrease in elastin accumulation as assessed by hot alkali treatment and transmission electron micrographs of the cell cultures. Tropoelastin formation was selectively decreased by rIL-1 beta relative to other proteins. Steady state levels of elastin mRNA were slightly decreased by rIL-1 beta at 5 pg/ml and markedly decreased by rIL-1 beta at 50 pg/ml or greater. The addition of indomethacin had no effect on rIL-1 beta-induced decreases in elastin mRNA levels. Inhibiting protein synthesis with cycloheximide blocked the effect of rIL-1 beta on elastin mRNA levels. The level of alpha 1(I) collagen mRNA was decreased by rIL-1 beta, but only at concentrations higher than that needed to induce a decrease in elastin mRNA. These data indicate that rIL-1 beta decreased steady state levels for elastin mRNA and elastin accumulation and can selectively regulate the accumulation of elastin and collagen.     

Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.     

A comparative analysis of the amino acid and cDNA sequences of bovine elastin a and chick elastin

A comparative analysis of the amino acid and cDNA sequences of bovine elastin a and chick elastin shows that there are considerable differences between these proteins. There is evidence that duplication of segments of DNA and gene rearrangement have occurred in the gene for chick elastin as compared with the gene for bovine elastin. The length of the polypeptide chain of elastin in both species is similar. Therefore, the duplications are compensated for by deletions in the gene for chick elastin.     

Primary structures of bovine elastin a, b, and c deduced from the sequences of cDNA clones

Nucleotide sequence analysis of cDNA clones for bovine elastin revealed the occurrence of three mRNAs for elastin in fetal calf nuchal ligament, encoding three forms of elastins (a, b, and c, of 747, 733, and 713 amino acid residues, respectively). These forms arise as the result of the presence, at a single position, of 102 additional nucleotides in the mRNA for elastin a and of 60 of these nucleotides in the mRNA for elastin b as compared to the mRNA for elastin c. As expected, most lysines occur in pairs, separated by two or three small amino acid residues. However, at two places, lysines occur in groups of three. The occurrence of a group of three lysines followed by a hydrophobic residue (lysine 400, 404, and 407) offers an explanation for the formation of lysinonorleucine. The alignment of amino acid sequences of porcine tropoelastin tryptic peptides with the sequence for bovine elastin a results in the ordering of these tryptic peptides. The analysis of the complete primary structures of elastin a, b, and c provides further insight into the structure-function relations of elastin.     

Accumulation and regulation of elastin in the rat uterus

The relative levels of elastin-specific mRNA were used as a measure of tropoelastin expression in uteri from pregnant Sprague-Dawley rats. The levels of elastin-specific mRNA were also correlated with values for net tropoelastin production and net deposition of mature, crosslinked elastin. The total content of uterine elastin increased throughout gestation, reaching maximal levels at Day 19 of gestation, which were three times those of nongravid tissue. Following involution, the elastin content decreased rapidly to near baseline values by 5 days postpartum. The content of soluble elastin, estimated using an enzyme-linked immunosorbent assay, paralleled in part the increase in elastin deposition and elastin mRNA levels. Uterine elastin metabolism appears to be unlike that in other elastic tissues, e.g., lung and large blood vessels. In most elastin containing tissues, the protein is synthesized during discrete developmental periods and is not readily degraded. However, uterine elastin is continuously expressed, and appears to be in a continual cycle of degradation and replacement.     

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.     

Creation of cross-linked electrospun isotypic-elastin fibers controlled cell-differentiation with new cross-linker

Effective application of elastin materials for vascular grafts in tissue engineering requires these materials to retain the elastic and biological properties of native elastin. To clarify the influence of soluble elastin isotypes on vascular smooth muscle cells (VSMCs), soluble elastin was prepared from insoluble elastin by hydrolysis with oxalic acid. Its fractions were separated and classified into three isotypes. Elastin retaining 2.25 mol% of cross-linked structures exhibited significant differentiation of VSMCs, which adhered to the elastin with contraction phenotypes similar to that of native elastin, causing proliferation to cease. This trend was more strongly demonstrated in cotton-like elastin fibers with a new cross-linker. The results suggest that elastin isotypes could be applied as new effective biomaterials for suppressing intimal hyperplasia in vascular grafts.     

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.     

Elastin peptides prepared from piscine and mammalian elastic tissues inhibit collagen‐induced platelet aggregation and stimulate migration and proliferation of human skin fibroblasts

We obtained pure elastin peptides from bovine ligamentum nuchae, porcine aorta, and bonito bulbus arteriosus. The inhibitory activity of these elastin peptides on platelet aggregation induced by collagen and the migratory and proliferative responsivenesses of human skin fibroblasts to these elastin peptides were examined. All of bonito, bovine, and porcine elastin peptides found to inhibit platelet aggregation, but bonito elastin peptides showed a higher inhibitory activity than bovine and porcine elastin peptides did. All elastin peptides enhanced the proliferation of fibroblasts 3.5‐ to 4.5‐fold at a concentration of 10 µg/ml. Bovine and porcine elastin peptides stimulated the migration of fibroblasts, with the optimal response occurring at 10^?1 µg/ml, while maximal response was at 10² µg/ml for bonito elastin peptides. Furthermore, pretreatment of fibroblasts by lactose depressed their ability to migrate in response to all elastin peptides, suggesting the involvement of elastin receptor in cell response. These results suggest that both mammalian and piscine elastin peptides can be applied as useful biomaterials in which elasticity, antithrombotic property, and the enhancement of cell migration and proliferation are required. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of sodium dodecyl sulphate with elastin polypeptides

The interaction that occurs between polypeptidic fragments of elastin and sodium dodecyl sulphate (SDS) as a model of natural amphiphilic substances has been studied by means of thermal concerration of the elastin fragments in the presence of detergents, by solubilization of a lipophilic dye, and by means of gel permeation chromatography. It was found that elastin polypeptides interact with SDS giving mixed micelles. This finding seems to be especially relevant in the tissues, revealing enhanced degradation of elastin and accumulation of lipophilic substances (e.g. in atheromatous plaques). In such tissues, elastin polypeptides formed could interfere with the formation of the normal elastic fibre by means of their interaction with amphiphilic substances.     

Influence of D-penicillamine on the formation of elastin and its cross-links.

The amount of insoluble elastin and its content of desmosine cross-links were investigated in aortas of chick embryos, to which D-penicillamine was administered on the 6th or 14th--16th day of incubation. D-Penicillamine was shown to alter the formation and maturation of elastin. Using lower doses (less than 50 mg) the weight of pooled aortic elastin is higher as compared with controls (related to 1 mg of elastin or to total weight of elastin). Increased isodesmosine:desmosine ratio in these samples indicates that this elastin is very young. On the other hand, a high dose of D-penicillamine (100 mg) decreased the content of elastin and also of its desmosine cross-links. The authors explain their findings by counteraction of two factors due to administration of penicillamine: the increased solubility of "insoluble elastin", and the decreased cross-link formation.     

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.     

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.     

Novel Angiotensin I-converting enzyme inhibitory peptides found in a thermolysin-treated elastin with antihypertensive activity

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC50 value of 531.6 μg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 μg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC50 value of 244 μM against ACE and may have potential use as a functional food.     

Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides.

3H-Labelled kappa-elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time-dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2.7 x 10(-10) M and 19,000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0.3 x 10(-9) M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could be inhibited by lactose and laminin but not by Arg-Gly-Asp-Ser(RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin-like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg-Gly-Asp-Ser(RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium-DTT extract was analysed by SDS-PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]-kE following nitrocellulose electro-transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]-kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin-derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres.     

Elastin production by cultured calf pulmonary artery endothelial cells

Calf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross-reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS-PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one-third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un-cross-linked, 70,000-dalton tropoelastin. The addition of beta-aminopropionitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross-linking of tropoelastin does not occur in culture. Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web-like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell-cell contact. Extracellular matrix remaining after solubilization of cellular material with Triton X-100 stained positive for fibronectin, but not for elastin.     

Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.