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.     

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.     

Recycling of the 67-kDa Elastin Binding Protein in Arterial Myocytes Is Imperative for Secretion of Tropoelastin

We have shown previously that the 67-kDa elastin binding protein (EBP) colocalizes intracellularly and extracellularly with tropoelastin in fetal sheep aorta, suggesting that these two proteins associate along the secretory pathway. Moreover, we have established that association with EBP protects tropoelastin from serine proteinases and from intracellular coacervation, and is necessary for its proper extracellular assembly. Since the production of tropoelastin by aortic smooth muscle cells (Ao SMC) exceeds production of the EBP, we speculated that this binding protein might recycle back into the cell, associating again with newly synthesized tropoelastin. In this report we labeled cultured Ao SMC externally with the F(ab′)₂ fragments of immunoglobulin which recognizes sheep EBP and followed trafficking of EBP by immunofluorescence and electron microscopy. Our results indicate that the majority of the EBP residing on the cell surface can be internalized to endocytic compartments (but not to lysosomes) and recycled back to the plasma membrane within 45-60 min. We have also determined that reagents disturbing pH of distinct endocytic compartments (chloroquine and bafilomycin A₁, but not ammonium chloride) arrest recycling of the EBP and, at the same time, strongly inhibit deposition of insoluble elastin in cultures of sheep Ao SMC and in organ cultures of chicken aorta. In contrast, neither chloroquine nor bafilomycin A₁ inhibit total protein synthesis or synthesis of tropoelastin. Our results suggest that the EBP serves as a reusable shuttle protein for tropoelastin and that its recycling is essential for effective deposition of insoluble elastin.     

Differential expression of aortic and lung elastin genes during chick embryogenesis

The ratios of tropoelastin b to a were measured in chick aorta and lung during embryogenesis. The rates of tropoelastin a and b synthesis were determined in short-term organ culture. The results demonstrated that in lung tissue the ratio of the two tropoelastins remained essentially constant. Each of the tropoelastins comprised 50% of the total elastin synthesis. In the aortic tissue, tropoelastin b represented 70% of the total elastin in the 11- to 13-day embryos and increased to 91% by Day 16. These observations seen in the organ culture system were paralleled in measurements of functional mRNAs coding for the two proteins. Measurements of functional tropoelastin mRNAs from both lung and aortic tissues were performed in a mRNA-dependent rabbit reticulocyte lysate system. Although the changes in the abundance of the tropoelastin mRNAs revealed the same trend as that seen in the organ culture data, the magnitude of the tropoelastin b to a ratio in the aortic organ culture was twice that determined in the cell-free translation of aortic mRNAs. The data obtained from both cell-free translations and organ culture experiments demonstrate that there is a differential expression of elastin genes during aorta development which is significantly different from that found in developing lung.     

Three-dimensional microstructural changes in murine abdominal aortic aneurysms quantified using immunofluorescent array tomography

This study investigated the spatial and temporal remodeling of blood vessel wall microarchitecture and cellular morphology during abdominal aortic aneurysm (AAA) development using immunofluorescent array tomography (IAT), a high-resolution three-dimensional (3D) microscopy technology, in the murine model. Infrarenal aortas of C57BL6 mice (N=20) were evaluated at 0, 7, and 28 days after elastase or heat-inactivated elastase perfusion. Custom algorithms quantified volume fractions (VF) of elastin, smooth muscle cell (SMC) actin, and adventitial collagen type I, as well as elastin thickness, elastin fragmentation, non-adventitial wall thickness, and nuclei amount. The 3D renderings depicted elastin and collagen type I degradation and SMC morphological changes. Elastin VF decreased 37.5% (p<0.01), thickness decreased 48.9%, and fragmentation increased 449.7% (p<0.001) over 28 days. SMC actin VF decreased 78.3% (p<0.001) from days 0 to 7 and increased 139.7% (p<0.05) from days 7 to 28. Non-adventitial wall thickness increased 61.1%, medial nuclei amount increased 159.1% (p<0.01), and adventitial collagen type I VF decreased 64.1% (p<0.001) over 28 days. IAT and custom image analysis algorithms have enabled robust quantification of vessel wall content, microstructure, and organization to help elucidate the dynamics of vascular remodeling during AAA development.     

The effect of beta-aminopropionitrile on elastin gene expression in smooth muscle cell cultures.

When beta-aminopropionitrile (BAPN) is added to neonatal rat aortic smooth muscle cell cultures there is a decrease in insoluble elastin accumulation with a concomitant increase in tropoelastin and tropoelastin fragments in the culture medium. The experiments described here examine the biological significance of this fragmentation. BAPN, as well as purified tropoelastin fragments isolated from spent medium of cells grown in the presence of BAPN, were added to cultures. A decrease in elastin mRNA was observed in cultures grown in the presence of BAPN and also in those cultures to which the purified tropoelastin moieties were added. These studies indicate that the inhibition of lysyl oxidase by BAPN prevents elastin crosslinking which results in an increase in tropoelastin moieties, thus leading to a down regulation of the steady state levels of elastin mRNA.     

67-kD elastin-binding protein is a protective "companion" of extracellular insoluble elastin and intracellular tropoelastin

The 67-kD elastin-binding protein (EBP) mediates cell adhesion to elastin and elastin fiber assembly, and it is similar, if not identical, to the 67-kD enzymatically inactive, alternatively spliced beta-galactosidase. The latter contains an elastin binding domain (S- GAL) homologous both to the aorta EBP and to NH2-terminal sequences of serine proteinases (Hinek, A., M. Rabinovitch, F. W. Keeley, and J. Callahan. 1993. J. Clin. Invest. 91:1198-1205). We now confirm the functional importance of this homology by showing that elastolytic activity of a representative serine elastase, porcine pancreatic elastase, was prevented by an antibody (anti-S-GAL) and by competing with purified EBP or S-GAL peptide. Immunohistochemistry of adult aorta indicates that the EBP exists as a permanent component of mature elastic fibers. This observation, together with the in vitro studies, suggests that the EBP could protect insoluble elastin from extracellular proteolysis and contribute to the extraordinary stability of this protein. Double immunolabeling of fetal lamb aorta with anti-S- GAL and antitropoelastin antibodies demonstrated, under light and electron microscopy, intracellular colocalization of the proteins in smooth muscle cells (SMC). Incubation of SMC with galactosugars to dissociate tropoelastin from EBP caused intracellular aggregation of tropoelastin. A tropoelastin/EBP complex was extracted from SMC lysates by coimmunoprecipitation and cross-linking, and its functional significance was addressed by showing that its dissociation by galactosugars caused degradation of tropoelastin by endogenous serine proteinase(s). This suggests that the EBP may also serve as a "companion" to intracellular tropoelastin, protecting this highly hydrophobic protein from self-aggregation and proteolytic degradation.     

Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at approximately 10(-8) M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucyl-phenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.     

Neuraminidase-1 is required for the normal assembly of elastic fibers

The assembly of elastic fibers in tissues that undergo repeated cycles of extension and recoil, such as the lungs and blood vessels, is dependent on the proper interaction and alignment of tropoelastin with a microfibrillar scaffold. Here, we describe in vivo histopathological effects of neuraminidase-1 (Neu1) deficiency on elastin assembly in the lungs and aorta of mice. These mice exhibited a tight-skin phenotype very similar to the Tsk mouse. Normal septation of Neu1-null mice did not occur in neonatal mice, resulting in enlarged alveoli that were maintained in adults. The abnormal development of elastic fibers was remarkable under electron microscopy and confirmed by the overlapping distribution of elastin, fibrillin-1, fibrillin-2, and fibulin-5 (Fib-5) by the light microscopy immunostainings. Fib-5 fibers appeared diffuse and unorganized around the alveolar walls and the apex of developing secondary septal crests. Fibrillin-2 deposition was also abnormal in neonatal and adult lungs. Dispersion of myofibroblasts appeared abnormal in developing lungs of Neu1-null mice, with a random distribution of myofibroblast around the alveolar walls, rather than concentrating at sites of elastin synthesis. The elastic lamellae in the aorta of the Neu1-null mice were thinner and separated by hypertrophic smooth muscle cells that were surrounded by an excess of the sialic acid-containing moieties. The concentration of elastin, as measure by desmosine levels, was significantly reduced in the aorta of Neu1-null mice. Message levels for tropoelastin and Fib-5 were normal, suggesting the elastic fiber defects in Neu1-null mice result from impaired extracellular assembly.     

Disruption of TGF-β signaling in smooth muscle cell prevents elastase-induced abdominal aortic aneurysm

Transforming growth factor-β (TGF-β) signaling has been significantly implicated in the pathogenesis of aneurysm, prominently the initiation and progression of abdominal aortic aneurysm (AAA). Vascular smooth muscle cell (SMC) is the principal resident cell in aortic wall and is essential for its structure and function. However, the role of TGF-β pathway in SMC for the formation of AAA remains unknown. Therefore, the goal of the present study was to investigate the effect of TGF-β pathway in SMC for AAA pathogenesis, by using a genetical smooth muscle-specific (SM-specific) TGF-βtype II receptor (Tgfbr2) disruption animal model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f and MyhCre.Tgfbr2^WT/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent AAA induction by infrarenal peri-adventitial application of elastase. Fourteen days after elastase treatment, the aortas were analyzed and indicated that disruption of 1 or 2 alleles of Tgfbr2 in SMC provided markedly step-wise protection from AAA formation. And elastin degradation, medial SMC loss, macrophage infiltration, and matrix metalloproteinases (MMP) expression were all significantly reduced in Tgfbr2 deletion mice. Our study demonstrated, for the first time, that the TGF-β signaling pathway in SMC plays a critical role in AAA and disruption can prevent the aneurysm formation.     

Inhibition of elastolysis by proteinase inhibitors from chick plasma and aorta

Chick plasma contains inhibitor(s) against trypsin and elastase which also appear to retard the degradation of tropoelastin by arterial tissue Chick aorta extracts also contain similar inhibitors against elastase and trypsin. Both levels of the plasma inhibitor(s) and inhibitor(s) extracted from thoracic aorta increase during early stages of growth and maturation. There is a three- to four-fold increase in the levels of the inhibitor(s) in chick plasma and aorta between one to four weeks after hatching. Of particular interest are the observations that the presence of the inhibitor(s) retards the conversion of soluble elastin (tropoelastin) to smaller elastin peptides. Subsequently, it is speculated that in addition to other vital roles, such proteinase inhibitors may also act in regulating elastogenesis and elastin fiber formation.     

Sequence and domain arrangements influence mechanical properties of elastin‐like polymeric elastomers

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self‐assembly that aligns lysine residues for crosslinking. As a result, both the full‐length monomer as well as elastin‐like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full‐length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self‐assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full‐length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 392–407, 2013.     

Pattern of accumulation of elastin and the level of mRNA for elastin in aortic tissue of growing chickens

Synthesis and accumulation of elastin in many elastic tissues begins in the last third of fetal development, reaches a maximum shortly after birth, and then declines rapidly. For the aorta of the chick and the pig and the ligamentum nuchae and lung of the sheep, it has been shown that increased levels of elastin production with fetal development are correlated with increased levels of elastin mRNA in the tissue, measured both by cell-free translation and by hybridization to cDNA probes. In this study we examine the relationship between insoluble elastin accumulation and message levels for tropoelastin in aortic tissue of chickens during posthatching development and growth. Whether evaluated by cell-free translation or by dot blot hybridization, steady state levels of tropoelastin message increase to a maximum at 2 weeks after hatching, and then fall rapidly with further development and growth. This pattern correlates well with production of insoluble elastin by the aorta, determined either by direct measurements of synthesis or by rate of accumulation of insoluble elastin. The data indicate that the major site of regulation of elastin production is pretranslational throughout the entire period of development and growth of the chicken aorta.     

Pro-inflammatory phenotype of COPD fibroblasts not compatible with repair in COPD lung

Chronic obstructive pulmonary disease (COPD) is characterized by loss of elastic fibres from small airways and alveolar walls, with the decrease in elastin increasing with disease severity. It is unclear why there is a lack of repair of elastic fibres. We have examined fibroblasts cultured from lung tissue from subjects with or without COPD to determine if the secretory profile explains lack of tissue repair. In this study, fibroblasts were cultured from lung parenchyma of patients with mild COPD [Global initiative for chronic Obstructive Lung Disease (GOLD) 1, n= 5], moderate to severe COPD (GOLD 2-3, n= 12) and controls (non-COPD, n= 5). Measurements were made of proliferation, senescence-associated β-galactosidase-1, mRNA expression of IL-6, IL-8, MMP-1, tropoelastin and versican, and protein levels for IL-6, IL-8, PGE(2,) tropoelastin, insoluble elastin, and versican. GOLD 2-3 fibroblasts proliferated more slowly (P < 0.01), had higher levels of senescence-associated β-galactosidase-1 (P < 0.001) than controls and showed significant increases in mRNA and/or protein for IL-6 (P < 0.05), IL-8 (P < 0.01), MMP-1 (P < 0.05), PGE(2) (P < 0.05), versican (P < 0.05) and tropoelastin (P < 0.05). mRNA expression and/or protein levels of tropoelastin (P < 0.01), versican (P < 0.05), IL-6 (P < 0.05) and IL-8 (P < 0.05) were negatively correlated with FEV1% of predicted. Insoluble elastin was not increased. In summary, fibroblasts from moderate to severe COPD subjects display a secretory phenotype with up-regulation of inflammatory molecules including the matrix proteoglycan versican, and increased soluble, but not insoluble, elastin. Versican inhibits assembly of tropoelastin into insoluble elastin and we conclude that the pro-inflammatory phenotype of COPD fibroblasts is not compatible with repair of elastic fibres.     

Modification of arterial elastin in vivo. Effects of age and diet on changes in the N-terminal amino acid content of aorta elastin

We have previously demonstrated that aorta elastin, a highly crosslinked protein, does not undergo turnover that is easily measured in vivo. Therefore, it was hypothesized that when proteolysis of elastin occurs, a positive increase in N-terminal amino acids should result. Such an increase would represent elastin-derived fragments held covalently in situ. A cyanate carbamylation procedure was used to estimate the changes in N-terminal amino acids in aorta elastin. To provide tissue for the studies, Japanese quail (3 weeks old) were fed diets with or without the addition of 1% cholesterol. It was found that, in normal birds, the number of N-terminal amino acid residues increased from two to approximately three residues per 800 total residues (or mole of tropoelastin) throughout sexual development (3 to 8 weeks, post-hatching), with little increase thereafter. In hypercholesterolemic birds, the rate of appearance of new N-terminal residues, particularly glutamine or glutamic acid, appeared enhanced throughout early development, but by sexual maturity the number of N-terminal amino acid residues in aorta elastin from cholesterol-fed birds was similar to that for the control birds. For each of the elastin samples analyzed, approximately one residue of glycine was recovered per 800 total residues. Other amino acids that predominated as N-terminal residues were serine, aspartic and glutamic acids.     

Elastin synthesis during perinatal lung development in the rat

The rate of soluble elastin synthesis was estimated in lung explants from rats of differing ages to better define periods in lung development important to the deposition of lung elastin. Lungs from rat pups at days 1, 3, 7, 9, 12, 15, and 21 post-parturition and from adult rats were incubated in a defined medium containing L-[3H]valine. Following incubation, labelled soluble elastin (tropoelastin) was separated from other soluble proteins by coacervation and electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The tropoelastin synthetic rate was then estimated after correcting for differences in recovery of radioactivity as tropoelastin and lung tissue L-[3H]valine specific activity. Maximal rates of elastin synthesis were observed in lung explants from 7-12-day-old rats. The rate of elastin synthesis during this period was 5-8-times the rate observed in adult rat lung (expressed per g of fresh lung) and represented approx. 2% of the total protein synthesis. Moreover, the values derived from lung explant culture for elastin synthesis were consistent with values for lung elastin deposition in the perinatal rat (5-10 micrograms elastin/h per g lung).     

Extracellular matrix-specific induction of elastogenic differentiation and maintenance of phenotypic stability in bovine ligament fibroblasts

We studied the process of elastogenic differentiation in the bovine ligamentum nuchae to assess the mechanisms that regulate elastin gene expression during development. Undifferentiated ( nonelastin - producing) ligament cells from early gestation animals initiate elastin synthesis when grown on an extracellular matrix (ECM) substratum prepared from late gestation ligamentum nuchae. ECM from ligaments of fetal calves younger than the time when elastin production occurs spontaneously in situ (i.e., beginning the last developmental trimester at approximately 180 d of gestation) does not stimulate elastin production in undifferentiated cells. Matrix-induced differentiation requires direct cell matrix interaction, is dependent upon cell proliferation after cell-matrix contact, and can be blocked selectively by incorporation of bromodeoxyuridine into the DNA of undifferentiated cells before (but not after) contact with inducing matrix. Quantitative analysis of elastin synthesis in young cells after matrix-induced differentiation indicates that the entire cell population is competent to respond to the matrix inducer, and continued synthesis of elastin after young cells are removed from the ECM substratum indicates that the phenotypic transition to elastin synthesis is stable and heritable. Although ligament cells do not require continuous contact with ECM to express the elastin phenotype, elastin synthesis is increased substantially when elastin-producing cells are grown on ligament matrix, suggesting that elastogenic differentiation is stabilized by ECM. The matrix substratum was also found to alter the distribution of tropoelastin between the medium and matrix cell layer. When grown on tissue culture plastic, ligament cells secrete greater than 80% of newly synthesized tropoelastin into the culture medium. When cultured on ECM, however, 50-70% of the newly synthesized tropoelastin remains associated with the cell layer and is cross-linked to form insoluble elastin as shown by the incorporation of radiolabeled lysine into desmosine.     

Sustained changes in lung expansion alter tropoelastin mRNA levels and elastin content in fetal sheep lungs

Our objective was to determine the effects of sustained alterations in fetal lung expansion on pulmonary elastin synthesis. In fetal sheep, lung expansion was either decreased between 111 and 131 days' gestation (term approximately 147 days) by tracheal drainage or increased for 2, 4, 7, or 10 days by tracheal obstruction, ending at 128 days' gestation. Lung tropoelastin mRNA levels were assessed by Northern blot analysis, total elastin content was measured biochemically, and staining of lung sections was used to assess the localization and form of elastic fibers. Tracheal obstruction significantly elevated pulmonary tropoelastin mRNA levels 2.5-fold at 2 days, but values were not different from controls at 4, 7, and 10 days; elastin content tended to be increased at all time points. A sustained decrease in lung expansion by tracheal drainage reduced pulmonary tropoelastin mRNA levels 2.5-fold; elastin content was also decreased compared with controls, and tissue localization was altered. Our results indicate that the degree of lung expansion in the fetus influences elastin synthesis, content, and tissue deposition.     

Radioimmunological identification of tropoelastin.

Antiserum was prepared in sheep against insoluble elastin isolated from embryonic-chick aortae. In an indirect immunoprecipitation test, the antiserum reacted quantitatively with small amounts of radioactively labelled purified tropoelastin prepared from embryonic-chick aortae. The antiserum did not cross-react with chick procollagen, and the antiserum uas used to identify radioactively labelled tropoelastin secreted by chick aorta cells in suspension culture.