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PubMed1999

Ultrastructural distribution of 36-kD microfibril-associated glycoprotein (MAGP-36) in human and bovine tissues.

We observed the ultrastructural distribution of MAGP-36 by immunoelectron microscopy in human and bovine tissues. MAGP-36 was present in microfibrils associated with tropoelastin in skin, aorta, and spleen. It was not detected in microfibrils from the ocular zonule and kidney mesangium that were not associated with tropoelastin. In skin, MAGP-36 was present in both early immature elastic fibers and mature elastic fibers. In mature elastic fibers, MAGP-36 was localized around amorphous elastic cores at the elastin-microfibril interface and in electron-dense bundles. Localization of MAGP-36 in elastic fibers coincided with the distribution of lysyl oxidase, an enzyme that plays a pivotal role in the deposition of tropoelastin. These findings suggest that MAGP-36 may be involved in elastogenesis.

Animals↗

PubMed1990

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

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

Actins↗

PubMed1987

Pulmonary fibroblasts: a model system for studying elastin synthesis.

Pulmonary interstitial fibroblasts were isolated from 4-day neonatal rat lung and cultured for varying periods of time after first passage. The ability of the cells to synthesize both soluble and insoluble elastin was examined by biochemical, immunological, and ultrastructural techniques. The cells synthesize a tropoelastin of 75,000 daltons which is cross-linked in the extracellular matrix to form sufficient amounts of insoluble elastin to be characterized directly by amino acid analysis. Although the cells produce a soluble form of elastin immediately after confluency and the deposition in the matrix begins shortly thereafter, the actual chemical definition of alkali-resistant elastin does not occur until 4 weeks after confluency. The apparent molecular weight of tropoelastin synthesized by the cells agrees with the molecular size of tropoelastin found in organ culture of neonatal lungs. The pulmonary fibroblast cultures provide an important model system for examining the regulation and response of elastin synthesis in lung interstitium.

Animals↗

PubMed1994

Conversion to an elastogenic phenotype by fetal hyaline chondrocytes is accompanied by altered expression of elastin-related macromolecules.

Elastic fibers are produced during fetal and neonatal periods, and by maturity elastin expression has ceased. Fetal bovine hyaline chondrocytes acquired an elastogenic phenotype within 24 hr after isolation from the tissue, even though the tissue does not produce elastic fibers or tropoelastin mRNA in vivo. By multiple parameters, hyaline chondrocytes produced elastic fibers that were indistinguishable from those made by elastic chondrocytes derived from fetal elastic ear cartilage. The levels of tropoelastin mRNA, secreted protein, and elastic fiber crosslinks as well as the immunostaining and ultrastructural appearance of elastic fibers produced by cultured hyaline chondrocytes were similar if not identical to those of cultured elastic chondrocytes. We also examined the expression of elastin-associated microfibrillar proteins. In intact hyaline cartilage, we did not detect mRNA for fibrillin 5 mRNA and saw only a relatively weak signal for fibrillin 15 mRNA. These microfibrillar products were expressed with tropoelastin in cultured hyaline chondrocytes as well as in intact elastic cartilage and cultured elastic chondrocytes, suggesting that fibrillin 5 and fibrillin 15 are required for elastic fiber formation. In contrast, the levels of microfibrillar-associated glycoprotein mRNA were decreased in both cell types relative to the high expression seen in vivo. These data indicate that conversion to the elastin phenotype includes induction or modulation of all known components of elastic fibers.

Animals↗

PubMed2003

Expression of elastic components in healthy and varicose veins.

This study evaluates possible changes in the synthesis/degradation of elastic components of the vein wall in an attempt to explain the development of varicosis. Healthy and varicose saphenous veins were subjected to immunohistochemical analysis using anti-elastin, anti-fibrillin-1, anti-elastase, anti-transforming growth factor (TGF)-beta and anti-latent TGFbeta binding protein (LTBP)-2 monoclonal antibodies. In situ hybridization was performed using specific probes for tropoelastin and fibrillin-1. In healthy veins, elastin and fibrillin-1 showed even, overlapping distribution patterns indicating their particular abundance in the adventitia and at the intima/media interface. The expression of tropoelastin and fibrillin-1 was high in smooth muscle cells bordering the elastic laminae. Elastin, fibrillin-1, and cells expressing fibrillin-1 and tropoelastin mRNA showed a patchy disorganized pattern, particularly in the proximal varicose segments of patients under 50 years of age. Enhanced elastase activity was noted in both control and varicose specimens from elderly subjects. Varicose veins specimens showed greater LTBP-2 and TGF expression. Both molecules were detected in the subendothelium and the media, particularly in areas of marked injury. Our findings suggest that the development of the varicose condition involves a restructuring of the elastic component of the vein wall, perhaps as a consequence of changes in the transcription mechanisms of muscle layer cells.

Adaptor Proteins, Signal Transducing↗

PubMed1975

A purification procedure for the isolation of homogeneous preparations of bovine aorta amine oxidase and a study of its lysyl oxidase activity.

It has been reported that bovine aorta amine oxidase oxidizes lysine residues in tropoelastin to allysine (Rucker, R.B. and O'Dell, B.L. (1971) Biochim. Biophys. Acta 235, 32-43). Pure bovine aorta amine oxidase was isolate by DEAE-cellulose, hydroxylapatite, Bio-Gel A-1.5 m and concanavalin A-Sepharose 4B chromatography. Enzymatic, chromatographic and immunochemical tests disclosed that pure bovine aorta amine oxidase was not a lysyl oxidase capable of oxidizing the lysine residues of tropoelastin to allysine; The bovine aorta amine oxidase preparation used by Rucker and O'Dell appears to have been contaminated with lysyl oxidase which is the emzyme that oxidizes some of the lysine residues in tropoelastin and tropocollagen to allysine.

Amino Acid Oxidoreductases↗

PubMed2005

Elastin.

Elastin is a key extracellular matrix protein that is critical to the elasticity and resilience of many vertebrate tissues including large arteries, lung, ligament, tendon, skin, and elastic cartilage. Tropoelastin associates with multiple tropoelastin molecules during the major phase of elastogenesis through coacervation, where this process is directed by the precise patterning of mostly alternating hydrophobic and hydrophilic sequences that dictate intermolecular alignment. Massively crosslinked arrays of tropoelastin (typically in association with microfibrils) contribute to tissue structural integrity and biomechanics through persistent flexibility, allowing for repeated stretch and relaxation cycles that critically depend on hydrated environments. Elastin sequences interact with multiple proteins found in or colocalized with microfibrils, and bind to elastogenic cell surface receptors. Knowledge of the major stages in elastin assembly has facilitated the construction of in vitro models of elastogenesis, leading to the identification of precise molecular regions that are critical to elastin-based protein interactions.

Animals↗

PubMed1987

The immunohistochemical localisation of microfibril-associated glycoprotein (MAGP) in elastic and non-elastic tissues.

We have previously identified the major antigen of elastin-associated microfibrils as a 31kD glycoprotein which we named microfibril-associated glycoprotein or MAGP. Affinity-purified antibodies to MAGP were shown to localise specifically to elastin-associated microfibrils in sections of bovine foetal nuchal ligament. In the present paper we compare the localisation of anti-MAGP antibodies and anti-tropoelastin antibodies in a range of bovine elastic and non-elastic tissues. The results show that anti-MAGP antibodies invariably localised to immuno-reactive elastic fibres, wherever they occurred. Extensive additional localisation was observed in a number of tissues. This extra distribution of anti-MAGP antibodies was found to correspond to those structures exhibiting the oxytalan histochemical staining reaction in tissues such as skin, periodontal ligament and ocular zonule. Since these oxytalan fibres have been shown to consist of 12 nm microfibrils which are morphologically similar to those of elastic fibres (and unpublished data from this laboratory confirm this conclusion), the results suggest that MAGP is a component of 12 nm microfibrils in both elastic and non-elastic tissues. Anti-tropoelastin antibodies did not localise to these oxytalan fibres, suggesting that tropoelastin is not a component of 12 nm microfibrils. MAGP was also detected in extracellular matrix regions of tissues such as skeletal muscle, Achilles tendon and spleen, suggesting that 12 nm microfibrils, containing one or more macromolecular constituents in common, make up an important structural system within the extracellular matrix in a wide range of elastic and non-elastic tissues.

Animals↗

PubMed2005

Fibulin-5 interacts with fibrillin-1 molecules and microfibrils.

Fibulin-5 plays an important role in elastic fibre formation in vivo. We have investigated the molecular interactions between fibulin-5 and components of fibrillin-rich microfibrils which form a template for elastin. Fibulin-5 interacted in a dose-dependent manner with a fibrillin-1 N-terminal sequence and with tropoelastin, but not with MAGP-1 (microfibril-associated glycoprotein-1) or decorin. Fibulin-5 did not inhibit interactions between fibrillin-1 N- and C-terminal fragments, or fibrillin-1 interactions with tropoelastin. Fibulin-5 may provide a link between tropoelastin and microfibrils in the pericellular space during elastic fibre assembly.

Contractile Proteins↗

PubMed1984

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.

Age Factors↗

PubMed1991

Regulation of elastin gene expression.

Recent isolation and characterization of cDNAs encompassing the full length of chicken, cow, and human elastin mRNA have led to the elucidation of the primary structure of the respective tropoelastins. Comparison of the tropoelastin from the different species has revealed that large segments of the sequence are conserved, but considerable variation also exists, ranging in extent from relatively small alterations, such as conservative amino acid substitutions, to large-scale deletions and insertions. Several distinct approaches have yielded compelling evidence of a single elastin gene per haploid genome. Analysis of the bovine and human elastin genes revealed that functionally distinct hydrophobic and cross-link domains of the protein are encoded in separate exons which alternate in the genes. The human gene contains 34 exons, the intron/exon ratio is unusually large (20:1), and the introns contain large amounts of repetitive sequences that may predispose to genetic instability. Comparison of the cDNA and genomic sequences has demonstrated that the primary transcript of both species is subject to considerable alternative splicing, which can account for the presence of multiple tropoelastin isoforms. It is likely that the conformation of elastin is, at least in part, that of a random coil, and therefore it might be expected that the stringency for conservation of the amino acid sequence would be less than that for other proteins with unique conformations. This suggests that functional elastin molecules that vary in their sequence and fitness may exist in the human population and be compatible with a normal life. Potentially though, these variations could have profound consequences on the properties of vital tissues found in the cardiovascular and pulmonary systems over the lifetime of the individual. Consequently, analysis of the structure of the elastin gene and its variation in what is regarded as the normal human population, rather than in those individuals with clearly heritable diseases, assumes greater importance. The 5'-flanking region of the gene is G + C rich and contains several SP-1 and AP2 binding sites, as well as putative glucocorticoid, cAMP, and TPA responsive elements, but no consensus TATA box or functional CAAT box. Primer extension and S1 mapping of the elastin mRNA indicated that transcription was initiated at multiple sites. Transfection experiments using promoter elements/reporter gene constructs demonstrated that the basic promoter element was found within region -128 to -1. In addition, three distinct up-regulatory and two down-regulatory regions were delineated. Taken together, these findings suggest that the regulation of elastin gene expression is complex and takes place at several levels.

Amino Acid Sequence↗

PubMed2004

Effects of UVR and UVR-induced cytokines on production of extracellular matrix proteins and proteases by dermal fibroblasts cultured in collagen gels%.

Synthesis of extracellular matrix (ECM) proteins and their degradation by matrix metalloproteinases (MMP) are part of the dermal remodeling resulting from chronic exposure of skin to ultraviolet radiation (UVR). We have compared two alternative mechanisms for these responses, namely, a direct mechanism in which UV-B or UV-A is absorbed by fibroblasts and an indirect mechanism in which cytokines, produced in skin in response to UVR, stimulate production of the ECM proteins and MMP. These studies were carried out on human dermal fibroblasts grown in contracted, free-floating 9 day old collagen gels as a dermal equivalent. Synthesis of tropoelastin, collagen, fibrillin, MMP-1, -2, -3 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 were measured. Tropoelastin, collagen and fibrillin levels were stable between days 4 and 10, and MMP and TIMP decreased by day 10. Neither UV-B (2.5-50 mJ/cm2) nor UV-A (2-12 J/cm2) altered synthesis of ECM proteins, but UV-A increased MMP-1 and -3 production. Tropoelastin synthesis increased in response to transforming growth factor-beta1 (5 ng/mL) treatment. Both interleukin-1beta and tumor necrosis factor-alpha (10 ng/mL) decreased fibrillin messenger RNA levels but increased MMP-1, -3 and -9 synthesis markedly. Collagen synthesis was not modulated by UV-B, UV-A or cytokine treatment. These results indicate that certain cytokines may have greater effects on production of ECM proteins and MMP than absorption of UV-B and UV-A by fibroblasts grown in dermal equivalents and suggest that the former pathway may play a role in the dermal remodeling in photoaged skin.

Cell Culture Techniques↗

PubMed1998

Degradation and repair of elastic fibers in rat lung interstitial fibroblast cultures.

BACKGROUND: Evidence from in vitro and in vivo studies indicates that damaged elastic fibers can be repaired. METHODS: Lipid interstitial pulmonary fibroblasts were cultured for 6 weeks. Cultures were then exposed to 25 microg of porcine pancreatic elastase and fixed in pairs (control, elastase-treated) immediately after exposure and at 1, 2, 3, 4, 7, 10, 14, and 22 days for ultrastructural examination. Elastin was also analyzed biochemically for resistance to hot alkali, an indicator of repair. Steady-state levels of tropoelastin and lysyl oxidase mRNA at 2, 4, and 7 days after elastase treatment were determined by Northern blot analysis. RESULTS: Immediately after exposure to elastase, damaged elastic fibers exhibited a frayed, porous appearance and a granular texture. Through day 4, fibers showed no evidence of repair. By day 7, the granular texture of damaged fibers was no longer evident and a gradual filling-in of porous areas appeared to be taking place. By 22 days, elastic fibers were indistinguishable from elastic fibers in control cultures. The ultrastructural changes were paralleled by changes in hot alkali resistance. Through day 4, there was no change in the level of hot alkali resistant elastin. Between day 4 and day 7, resistance to hot alkali increased sharply and continued to increase at a slower rate, reaching 84% of the control level by day 22. Steady-state levels of tropoelastin and lysyl oxidase mRNA showed no increase over control levels at 2, 4, and 7 days after elastase treatment. CONCLUSIONS: Elastic fibers synthesized by lipid interstitial pulmonary fibroblasts in culture were repaired after damage by elastase. This type of repair may have relevance to the prevention of pathological conditions, such as emphysema.

Animals↗

PubMed1989

Structure of the elastin gene and alternative splicing of elastin mRNA: implications for human disease.

The protein elastin is largely responsible for the elastic properties of vertebrate lungs, large blood vessels, and skin. The structure of the human, bovine, and chick elastin gene and protein monomer, tropoelastin, has recently been elucidated by using techniques of molecular biology. Extensive homology of amino acid sequence exists among the mammalian species and there is in addition strong conservation of nucleotide sequences in the 3' untranslated region of the gene. The translated exons are small and embedded in large expanses of introns. Sequences coding for the hydrophobic regions, responsible for the elastic properties of the molecule, and the alanine-lysine rich regions, responsible for crosslink formation between molecules, reside in separate exons and alternate for the most part in the elastin gene. S1 analyses and sequence analysis of cDNA and genomic clones have indicated that there is substantial alternative splicing of the primary elastin transcript. Variations in the structure of mRNAs resulting from alternative splicing could explain the existence of the multiple forms of tropoelastin observed electrophoretically in several species. Different kinds of splicing patterns could occur in human populations and may contribute to aging and pathological situations in the cardiovascular and pulmonary systems.

Aging↗

PubMed1986

Role of plasma and serum proteases in the degradation of elastin.

Accelerated proteolysis of tropoelastin and elastin occurs in the major arteries of chicks fed copper-deficient diets. Signs of elastin degradation are not obvious in normal arteries of copper-supplemented chicks. It is proposed that the sources of proteases that effect elastin degradation are from plasma and serum. Both calcium-dependent proteases and kallikrein were effective in degrading tropoelastin and partially crosslinked insoluble elastin into peptides similar to those detected in aortic extracts from copper-deficient chicks. As dietary copper deficiency progresses it is also possible to detect elastin peptides in plasma.

Animals↗

PubMed1988

Chemotaxis of fibroblasts toward nonapeptide of elastin.

Bovine ligamentum fibroblasts, which produce elastin, migrate towards a positive chemical gradient of human platelet-derived growth factor and of the tropoelastin repeat hexapeptide Val-Gly-Val-Ala-Pro-Gly, as previously shown. They are also responsive to two permutations of a nonapeptide that repeats in tropoelastin, i.e., Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly and Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro. Concentration curves and checkerboard assays prove that the nonapeptides are chemoattractants. The component pentapeptide, Gly-Phe-Gly-Val-Gly, is chemotactic, while the component tetrapeptide Ala-Gly-Val-Pro is not. The hexapeptide competitively suppresses the nonapeptide chemotaxis suggesting the involvement of a common cell receptor. The results support the concept that elastin has multiple cell recognition sites as measured by the chemotactic response and that among the hydrophobic repeating sequences of elastin chemotacticity is selectively and multiply localized.

Animals↗

PubMed1978

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 extracts. 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.

Animals↗

PubMed2005

Cellular interactions with elastin.

Elastin is a key structural component of the extracellular matrix. Tropoelastin is the soluble precursor of elastin. In addition to providing elastic recoil to various tissues such as the aorta and lung, elastin, tropoelastin and elastin degradation products are able to influence cell function and promote cellular responses. These responses include chemotaxis, proliferation and cell adhesion. The interaction of elastin products with cells has been attributed to the elastin receptor. However, additional cell-surface receptors have also been identified. These include G protein-coupled receptors and integrins. The potential roles of these receptors in cell-elastin interactions, with particular focus on elastin formation are discussed.

Amino Acid Sequence↗