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PubMed2000

Hydrostatic pressure stimulates synthesis of elastin in cultured optic nerve head astrocytes.

Elastin is a major component of the extracellular matrix (ECM) of the lamina cribrosa in the optic nerve head in humans and nonhuman primates. The lamina cribrosa appears to be the site of damage to the retinal ganglion cell axons in glaucomatous optic neuropathy, characterized in many patients by elevated intraocular pressure (IOP). Type 1B astrocytes are the major cell type in the lamina, synthesize elastic fibers during development, express increased elastin mRNA, and synthesize abnormal elastin in glaucoma. In this study, we determined the effect of elevated hydrostatic pressure on the synthesis of elastin by type 1B astrocytes in culture. Type 1B astrocytes were exposed to gradients of hydrostatic pressure and tested for proliferation, morphology, synthesis, and deposition of elastin. Trichloroacetic acid (TCA) and immunoprecipitation of radiolabeled protein determined total new protein and elastin synthesis. Proteins from the conditioned media were analyzed by Western blot. Levels of elastin mRNA were determined by in situ hybridization. Cell proliferation increased approximately 2-fold after exposure to pressure for one day, approximately 5-fold after 3 and 5 days of exposure to pressure. Confocal and electron microscopic cytochemistry showed a marked increase in intracellular elastin in astrocytes exposed to pressure, as compared with controls. Intracellular elastin was associated with the RER-Golgi region and with the cytoskeleton. Total protein and elastin synthesis increased significantly (P < 0.05) at 3- and 5-day exposure to pressure, as well as the level of elastin mRNA. Elastin protein in the media increased with the level of pressure. These results indicate that hydrostatic pressure stimulates type 1B astrocytes to synthesize and secrete soluble elastin into the media. In glaucoma, type 1B astrocytes may respond to IOP-related stress with increased expression of elastin and formation of elastotic fibers leading to loss of elasticity and tissue remodeling.

Adolescent↗
PubMed1993

Effects of heparin and other glycosaminoglycans on elastin production by cultured neonatal rat lung fibroblasts.

Proteoglycans are important structural elements of the extracellular matrix, and may contribute to the dynamic architecture of the lung and also influence pulmonary gas and solute exchange. The potential for proteoglycans and glycosaminoglycans to modulate the synthesis and deposition of elastin, another important extracellular matrix component of the lung, has not been established. Therefore the effects of glycosaminoglycans on the steady-state level of elastin mRNA, the incorporation of [3H]valine into tropoelastin, the distribution of soluble elastin in the medium and cell layer, and insoluble elastin deposition have been examined using cultured neonatal rat lung fibroblasts. Heparin decreases the soluble elastin content of the culture medium while increasing the soluble elastin content of the cell layer. This altered partitioning of soluble elastin is associated with an increase in steady-state elastin mRNA and an increase in the deposition of insoluble elastin in the extracellular matrix. Some of these effects may result from the binding of heparin to soluble elastin at physiological concentrations of NaCl. The galactosamine-containing glycosaminoglycans, chondroitin sulfate and dermatan sulfate, differ from heparin in that they increase the quantity of soluble elastin in the culture medium and decrease the deposition of insoluble elastin in the extracellular matrix. Proteoglycans, which are present in most elastic tissues, may participate in the regulation of elastin synthesis and deposition during periods of new elastin formation.

Aminopropionitrile↗
PubMed1994

Basic fibroblast growth factor decreases elastin production by neonatal rat lung fibroblasts.

During pulmonary development, there is a burst in elastin synthesis by interstitial fibroblasts coincident with the period of alveolar septal elongation. Little is known about the regulation of elastin synthesis by these cells, although several endocrine and paracrine factors influence lung fibroblast elastin production. Because alveolar septal elongation is accompanied by a decrease in capillary endothelial cell mitosis, we have hypothesized that a reduction in basic fibroblast growth factor (bFGF), an endothelial cell mitogen, may occur concomitantly with an increase in elastin synthesis. This temporal relationship suggests that bFGF may influence elastin production by interstitial fibroblasts. Therefore, we have examined the effects of bFGF on elastin production by postconfluent, serum-free cultures of lipid interstitial fibroblasts (LF). Elastin production was quantitated by analyzing the incorporation of 3H-valine into the soluble elastin precursor tropoelastin (TE). Exogenous bFGF decreased the quantity of newly synthesized TE in the culture media and cell layers of LF. The level of newly synthesized TE in the media was decreased to 36% and 48% of the unexposed control when LF were exposed for 48 h to 10 or 75 ng/ml bFGF, respectively. Northern analysis demonstrated that the decrease in TE was accompanied by a similar decrease in elastin mRNA. Transient transfection experiments using an elastin promoter/CAT gene construct demonstrated that bFGF exposure decreased elastin promoter activity. These results suggest that bFGF decreases elastin transcription. Exposure to an anti-bFGF antibody neutralized endogenous bFGF and increased soluble elastin production by LF. Our studies indicate that exogenous and endogenous bFGF can suppress elastin production by LF. A similar effect may occur in the intact lung during development or chronic inflammation.

Analysis of Variance↗
PubMed1973

The interaction of serum and arterial lipoproteins with elastin of the arterial intima and its role in the lipid accumulation in atherosclerotic plaques.

Arterial elastin appears to be a proteinlipid complex with the lipid component being bound to elastin peptide groups. In atherosclerotic lesions the lipid content of elastin increases progressively with increasing severity of atherosclerosis. The increases in the lipid content of plaque elastin are mainly due to large increases in cholesterol with about 80% of the cholesterol being cholesterol ester. This deposition of cholesterol in elastin accounts for a substantial part of the total cholesterol accumulation in atherosclerotic lesions of all stages. The present in vitro study suggests that the mechanism involved in the deposition of lipids in arterial elastin may be an interaction of the elastin protein with serum or arterial low density or very low density lipoproteins (LDL and VLDL) resulting in a transfer of lipids, but not of lipoprotein protein to the elastin. No significant lipid transfer occurred from the high density lipoproteins or chylomicrons. The amount of lipid taken up by plaque elastin was strikingly higher than by normal elastin and consisted mainly of cholesterol with over 80% of the cholesterol being cholesterol ester. The precondition for the lipid accumulation in plaque elastin appeared to be an altered amino acid composition of the elastin protein consisting of an increase in polar amino acids and a reduction in cross-linking amino acids. Subsequent treatment of lipoprotein-incubated arterial elastin with hot alkali and apolipoproteins did not reverse the binding of lipoprotein lipid to diseased elastin.

Amino Acids↗
PubMed1976

Amidated carboxyl groups in elastin.

Dicarboxylic amino acids constitute the most numerous residues of insoluble elastin in which are potentially ionizable in the physiological range of pH. These residues are essential in facilitating productive electrostatic interaction between elastase and elastin. The present study has investigated the possibility that the glutamic and aspartic acid residues of elastin are amidated. Acid-labile amide-bound ammonia of elastin was quantitated after hydrolysis of the insoluble protein with 2 M HC1 by incubating aliquots of microdistilled hydrolysates with glutamate dehydrogenase, excess alpha-ketoglutarate, and reduced nicotinamide adenine dinucleotide and measuring the resultant decrease in A340 due to oxidation of the dinucleotide cofactor. It was found that ligament elastin purified by repeated autoclaving contains approximately 2.29 mumol of acid-labile amide nitrogen per 10 mg of protein, a value equivalent to approximately 70% of the total number of dicarboxylic amino acid residues. Independent analysis of the amide content was obtained by amino acid analysis of an esterified and reduced elastin sample in which the free dicarboxylic amino acid residues had been converted to the corresponding alcohol derivatives. This analysis indicated that autoclaved ligament elastin contains approximately 18 glutamine, 3 asparagine, 4 glutamic acid and 5 aspartic acid residues per 1000 residues, in good agreement with the analysis of total acid-labile ammonia. The esterified and reduced elastin derivative was nearly inert as an elastase substrate, consistent with a lack of free dicarboxylic amino acid residues. However, addition of sodium dodecyl sulfate to this elastin derivative restores enzyme-substrate charge complementarity, and the elastin-ligand complex was readily hydrolyzed by elastase at the fully stimulated rate, emphasizing the control such ligands can exert in elastolysis. The amide bonds of elastin were found to be significantly more resistant to hydrolysis by 0.1 M NaOH at 98 degrees C than were those of lysozyme or free amidated amino acids. The finding that most of dicarboxylic amino acid residues of elastin exist at neutral amides further emphasizes the apolar character of elastin and has bearing upon the metabolic susceptibility, ligand-binding ability and structural aspects of this connective tissue protein.

Amides↗
PubMed2006

Idiotypic and anti-idiotypic elastin autoantibodies: Implications for IVIg and pregnancy loss.

PROBLEM: The aim of this study was to investigate anti-elastin and anti-anti-elastin autoantibodies in intravenous immunoglobulin (IVIg) lots as an attempt to further explain the effect of IVIg in recurrent pregnancy loss (RPL). METHOD OF STUDY: Serum samples of 10 female patients with RPL and 10 healthy subjects were tested for anti-elastin autoantibodies and used in competitive inhibition studies. A total of 44 IVIg lots (ZLB Behring, Switzerland) were tested for anti-elastin and anti-anti-elastin idiotypes. One way analysis of variance (ANOVA) and Least Significant Difference (LSD method) were used for statistical analysis of differences between the lots. RESULTS: Serum anti-elastin IgG autoantibodies were significantly higher in the study group, compared to the controls. In all lots anti-elastin IgG antibodies were identified. All lots (except two of them) showed similar dose-dependent inhibition of serum anti-elastin activity by anti-elastin anti-idiotypes in IVIg. CONCLUSIONS: Anti-elastin IgG autoantibodies were increased in patients with RPL - a finding which needs further explanation. Anti-elastin and anti-anti-elastin idiotypes were identified in different IVIg lots. The presence in IVIg of anti-idiotypes against anti-elastin autoantibodies from patients' sera could be an additional mechanism of the beneficial effect of IVIg in reproductive failure.

Abortion, Habitual↗
PubMed1997

Ascorbate differentially regulates elastin and collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts by pretranslational mechanisms.

Ascorbate contributes to several metabolic processes including efficient hydroxylation of hydroxyproline in elastin, collagen, and proteins with collagenous domains, yet hydroxyproline in elastin has no known function. Prolyl hydroxylation is essential for efficient collagen production; in contrast, ascorbate has been shown to decrease elastin accumulation in vitro and to alter morphology of elastic tissues in vivo. Ascorbate doses that maximally stimulated collagen production (10-200 microM) antagonized elastin biosynthesis in vascular smooth muscle cells and skin fibroblasts, depending on a combination of dose and exposure time. Diminished elastin production paralleled reduced elastin mRNA levels, while collagen I and III mRNAs levels increased. We compared the stability of mRNAs for elastin and collagen I with a constitutive gene after ascorbate supplementation or withdrawal. Ascorbate decreased elastin mRNA stability, while collagen I mRNA was stabilized to a much greater extent. Ascorbate withdrawal decreased collagen I mRNA stability markedly (4.9-fold), while elastin mRNA became more stable. Transcription of elastin was reduced 72% by ascorbate exposure. Differential effects of ascorbic acid on collagen I and elastin mRNA abundance result from the combined, marked stabilization of collagen mRNA, the lesser stability of elastin mRNA, and the significant repression of elastin gene transcription.

Animals↗
PubMed1992

Relation of serum elastin peptide concentration to age, FEV1, smoking habits, alcohol consumption, and protease inhibitor phenotype: an epidemiological study in working men.

BACKGROUND: In clinical investigations elastin peptide concentration has been proposed as one potential marker of lung elastin degradation. No epidemiological study has yet confirmed this hypothesis. METHODS: The relation of elastin peptide concentration to some factors closely related to pulmonary emphysema (age, smoking habits, FEV1 alpha protease inhibitor (PI) phenotype) and to alcohol consumption was examined in an epidemiological study of 310 working men. The elastin peptides used for obtaining antibodies and as reference in an ELISA assay were prepared from chemically hydrolysed elastin. RESULTS: The elastin peptide concentration significantly decreased with age from 2.92 (1.54) micrograms/ml among subjects younger than 30 years to 2.18 (1.14) micrograms/ml among subjects older than 50. Elastin peptide concentration did not differ with smoking habits and was clearly unrelated to FEV1. A lower elastin peptide concentration was observed in all groups of subjects with a protease inhibitor phenotype other than PI MM (PI FM, IM, MP, MS, MZ, and S phenotypes). CONCLUSIONS: The results cast doubts on the usefulness of the elastin peptide concentration as a marker of lung destruction in middle aged, predominantly healthy men. Blood elastin peptide concentration may reflect both elastin degradation and resynthesis. The results of this analysis suggest that several factors (age, alcohol consumption, non-PI MM phenotype) may be associated with decreased resynthesis of lung elastin. Further studies, conducted in various age groups and including estimates of the degree of lung destruction, are needed to unravel the mechanisms underlying lysis and resynthesis of lung elastin.

Adult↗
PubMed1993

Retinoic acid increases elastin in neonatal rat lung fibroblast cultures.

The factors that regulate elastin synthesis during pulmonary alveolar septal formation have not been identified. Because maximal alveolar elastin synthesis occurs over a relatively brief period (postnatal days 4-14 in the rat), we hypothesized that changes in the local concentrations of factors that regulate elastin synthesis may precede or accompany this period. Because pulmonary retinoid stores decline just before the fourth postnatal day, we also hypothesized that this decline could be accompanied by the utilization of retinoic acid, one of the most biologically active retinoids, in a regulatory process that increases elastin synthesis. If these hypotheses are correct, then retinoic acid should increase elastin synthesis by pulmonary cells. Therefore, cultures of neonatal rat lung fibroblasts were exposed to retinoic acid, and elastin production was quantitated. Retinoic acid produced a two- to threefold increase in the steady-state level of elastin mRNA, in soluble elastin, and in insoluble elastin. The transcriptional initiation rate of the elastin gene was 1.8-fold higher in nuclei that were isolated from retinoic acid-treated cells than in nuclei that were isolated from control cells. This indicates that the increase in steady-state elastin mRNA results, at least partially, from an increase in elastin transcription. Lung fibroblasts that were isolated from 8-day-old rats, but not cultured, contained retinoic acid. These findings suggest that retinoic acid is a potential regulator of elastin synthesis in developing pulmonary alveoli.

Animals↗
PubMed1989

Elastin degradation by human alveolar macrophages. A prominent role of metalloproteinase activity.

Macrophages are thought to play an important role in the turnover of extracellular matrix, but the capacity of human macrophages to degrade elastin, and the elastolytic mechanisms of these cells, have been controversial. Particular difficulty has been encountered in efforts to establish whether human macrophages secrete a metalloelastase that is analogous to the enzyme secreted by rodent macrophages. We studied elastin degradation by human alveolar macrophages cultured directly in contact with radiolabeled elastin using media containing 10% fetal bovine serum, and for comparison performed parallel studies of P388D1 murine macrophagelike cells that are known to secrete metalloelastase. With both cell types, we observed elastin degradation and the following: (1) direct contact between the cells and elastin substrate was required for elastin degradation; (2) elastin degradation was inhibited by the tissue inhibitor of metalloproteinases, but minimally or not at all by inhibitors of cysteine proteinases (E-64, CBZ-phe-phe-CHN2, CBZ-phe-ala-CHN2, and cystatin C), or by the serine proteinase inhibitor eglin-c; (3) elastin degradation increased sharply after the cells were in contact with elastin for 24 h, and required new protein synthesis as indicated by sensitivity to cycloheximide; (4) inclusion of dexamethasone (10(-6) to 10(-8) M) in the cultures led to decreased elastin degradation. Also, with both cell types, elastin degradation occurred despite the finding that cell-conditioned media did not contain elastase activity and could inhibit P388D1-derived metalloproteinase elastase. These results indicate a prominent role for metalloproteinase activity in elastin degradation by both human and murine macrophages and support the concept that events at the cell-substrate interface are critically important to macrophage-mediated elastin degradation.

Animals↗
PubMed1988

Altered elastin and collagen synthesis associated with progressive pulmonary hypertension induced by monocrotaline. A biochemical and ultrastructural study.

Changes in elastin and collagen synthesis in the pulmonary artery wall, assessed both biochemically and ultrastructurally, were related to the development of progressive pulmonary hypertension induced by the toxin monocrotaline. Male Sprague-Dawley rats (200 to 225 gm) were injected subcutaneously in the hind flank with either monocrotaline (60 mg/kg) or an equivalent volume of saline and studied 8, 16 and 28 days later. At each time point, the right ventricle and left ventricle with septum were separated and weighed to follow the development of right ventricular hypertrophy. The hilar pulmonary artery was assessed by light microscopy for medial hypertrophy and by electron microscopy for changes in the endothelium, subendothelium and media. The mainstem pulmonary artery was used to determine synthesis of elastin and collagen by in vitro incorporation of [14C]proline into nonelastin, [14C]hydroxyproline into collagen, and [3H]valine into cyanogen bromide-insoluble elastin. In addition, total content of insoluble elastin was determined by weight of the residue after cyanogen bromide digestion and of collagen by total hydroxyproline content in sodium dodecyl sulfate and cyanogen bromide extracts. Eight days after monocrotaline injection, there was pulmonary artery endothelial swelling and a significant decrease in the number of myoendothelial junctions (p less than 0.05) associated with a decreased proportion of amorphous elastin in the media (p less than 0.01). Sixteen days after monocrotaline injection, a decrease in the proportion of elastin in the media was still evident (p less than 0.01) despite an apparent increase in insoluble elastin synthesis. Moreover, the amorphous elastin was distributed preferentially in small islands rather than in laminae (p less than 0.05). Twenty-eight days after monocrotaline injection, medial and right ventricular hypertrophy had developed (p less than 0.05 and p less than 0.01, respectively). At the same time, there was a striking increase in both insoluble elastin synthesis and total insoluble elastin content (p less than 0.01 for both) and an increase in collagen synthesis and total collagen content (p less than 0.05 for both). In addition, the ratio of insoluble elastin synthesis to collagen synthesis was greater than in controls (p less than 0.01), whereas the ratio of total insoluble elastin to total collagen did not change. On ultrastructural analysis, the proportion of amorphous elastin in the vessel wall relative to other elements remained low (p less than 0.01) and was distributed throughout the media as increased numbers of small islands (p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

Differential expression of elastin and alpha 1(I) collagen mRNA in mice with bleomycin-induced pulmonary fibrosis.

Interstitial pulmonary fibrosis is characterized by increased production of connective tissue components, including collagen and elastin. The role of elastin turnover in pulmonary fibrosis is not clear, and it is not known whether elastin and collagen are regulated separately or together during the inflammatory process. Mice with bleomycin-induced pulmonary fibrosis were investigated to determine the temporal and spatial changes in localization of elastin mRNA expression, as well as to compare elastin mRNA expression with that of alpha 1(I) collagen mRNA expression. In control (saline-treated) lungs, elastin mRNA was detected by in situ hybridization in arterial walls. No signal was found in alveolar or airway walls or in pleura; alpha 1(I) collagen mRNA was detected in the tissue underlying the airway epithelium. An increase in elastin mRNA expression in muscular arteries was observed 3 days after bleomycin instillation. Expression was also seen in the adventitia of terminal airways and adjacent small blood vessels. Expression of alpha 1(I) collagen mRNA increased in the tissue underlying the airway epithelium. In the pleura, alpha 1(I) collagen mRNA expression was found, although no pleural thickening was evident. The alpha 1(I) collagen mRNA expression was particularly increased in the adventitia of terminal airways and in associated small blood vessels. Obvious in areas of fibrosis, elastin mRNA expression was occasionally increased in the pleura and airway wall 7 days after bleomycin treatment; alpha 1(I) collagen mRNA expression was generally stronger than elastin mRNA expression in areas of fibrosis and was frequently intense in the adventitia of airways and associated blood vessels. The fibrotic areas showed increased elastin mRNA expression 14 and 30 days after bleomycin treatment. The arteries in fibrotic areas showed normal elastin mRNA levels. In the areas of fibrosis and in the adventitia of airways and adjacent blood vessels, alpha 1(I) collagen mRNA expression was very high. In conclusion, lung elastin biosynthesis is markedly altered by bleomycin treatment. The localization and intensity of elastin mRNA expression is different from the expression of alpha 1(I) collagen mRNA.

Animals↗
PubMed1995

Skin elastic fibres: regulation of human elastin promoter activity in transgenic mice.

Elastic fibres form an extracellular network which provides elasticity and resilience to tissues such as the skin. To study the regulation of human elastin gene expression, we have developed a line of transgenic mice which harbour 5.2 kb of human elastin gene promoter region in their genome. This promoter is linked to the chloramphenicol acetyltransferase (CAT) reporter gene which allows determination of the expression of human elastin promoter in different tissues. The highest CAT activity was found in the lungs and aorta, tissues rich in elastin, while lower levels were detected in a variety of other tissues, including skin. Assay of CAT activity in the lungs of fetal and newborn animals revealed high activity which progressively declined during the postnatal period up to six months. Thus, there was evidence of tissue-specific and developmentally regulated expression of the human elastin promoter activity in these mice. These animals were then used to examine the expression of the elastin gene by a variety of factors which have previously shown to alter elastin gene expression, as determined at the mRNA or protein levels. First, injection of transforming growth factor beta 1 (100 ng) subcutaneously into the transgenic animals resulted in a time-dependent elevation of the promoter activity up to 10-fold after a single injection. Secondly, enhancement of the human elastin promoter activity by interleukin 1 beta injected subcutaneously resulted in an approximately 10-fold elevation of the CAT activity. Finally, subcutaneous injection of these animals with triamcinolone acetonide or dexamethasone, two glucocorticosteroids in clinical use, resulted in marked enhancement of human elastin promoter activity. Similar changes were noted in fibroblast cultures established from the transgenic animals. These data indicate that the 5.2 kb upstream segment of the human elastin gene contains cis-elements which allow tissue-specific and developmentally regulated expression of the human elastin promoter. Furthermore, this segment of the gene contains responsive elements to a variety of cytokines and pharmacological agents. Collectively, these data indicate that elastin gene expression in the skin in vivo can be regulated at the transcriptional level.

Animals↗
PubMed1996

Interactions of elastin and aorta with sugars in vitro and their effects on biochemical and physical properties.

Stiffening of blood vessel walls occurs in the early stages of atherosclerosis, and this process is known to occur earlier in diabetic subjects. The effect could be due, in part, to glycation. Although collagen is responsible for ensuring the ultimate tensile strength of the tissue, elastin largely determines the compliance of the vessel wall in the normal physiological range of pressures and this appears to be closely matched to haemodynamic requirements. Changes in elastin are therefore likely to affect optimal function of the tissue. We have investigated the susceptibility of elastin to glycation and effects of glycation on its mechanical and physicochemical properties. We found that purified elastin and a collagen-elastin preparation from the porcine thoracic aorta rapidly incorporated glucose and ribose, the extent increasing linearly with increasing concentration and reaching a maximum after 7 days at 37 degrees C. Biochemical analysis showed that about one of the five lysines available per elastin monomer was glycated after 12 days incubation at a sugar concentration of 250 mmol/l. In long-term incubations glycation was associated with the appearance of the advanced glycation end products, the fluorescent cross-link pentosidine and the non-fluorescent putative cross-link NFC-1. In both purified elastin and the whole elastin-collagen matrix the slope of the force-extension curve increased significantly with glycation. The greatest increase in stiffness was observed in the elastin-collagen preparation after ribose incubation (250 mmol/l for 1 month), where the slope, at large strain, increased by 56 +/- 19% (mean +/- SD, n = 12). The diameter of the tissue at 1 N force also changed: for elastin there was an increase in length of approximately 5%, but for the elastin-collagen there was a decrease of similar magnitude indicating that glycation introduces differential strains within the fibrous protein matrix. Potentiometric titration demonstrated that glycation was associated both with loss of basic groups and shifts in pK of the acidic groups, which indicated changes in the environment of the charge groups due to conformational rearrangements. Changes in ion binding were dependent on pH, and were consistent with a reduction in effective anionic charge. Calcium binding to elastin was increased at acid pH, but decreased at higher pH. We suggest that these effects are not only due to changes in the charge profile, but also in the conformation of the molecule resulting from glycation of the charged lysine and arginine side-chain residues.

Amino Acid Sequence↗
PubMed1997

Effect of age on the vasodilatory action of elastin peptides.

We have recently shown, on young adult rat aorta rings, that elastin peptides induce a dose and endothelium-dependent vasodilation mediated by the 67 kDa subunit of the high affinity elastin-laminin receptor and, at least in part, by EDRF (NO). Here we have studied the effects of elastin peptides at circulating concentrations and below, on noradrenaline-contracted rat aortic rings, as a function of age. First, we have observed that, unlike 2-month-old (2M), 4-6-month-old (4M) and 12-month-old (12M) rat aorta rings, 30-month-old (30M) rat aorta rings were unable to maintain their contraction in long lasting experiments. Secondly, elastin peptides at physiological circulating concentrations (10(-6)-10(-3) mg/ml) induce a dose-dependent vasodilation on 4M rings. By contrast, only higher elastin peptide concentrations (10(-3) mg/ml) were effective on 12M rings, whereas rings from both younger (2M) and older animals (30M) did not respond to elastin peptides. Finally, using lactose and laminin as inhibitors, we have demonstrated that elastin peptide-induced vasodilation on 4M and 12M rings is mediated by the 67 kDa subunit of the elastin-laminin receptor. These experiments suggest that the functional availability of the 67 kDa subunit of the elastin-laminin receptor changes with age. It could be hypothesized that in young animals (0-2M) the reusable shuttle role recently demonstrated for the 67 kDa receptor subunit during elastic fiber formation leads to a major decrease in its availability for signal transduction. On the contrary, in adult animals. (4-12M), when developmental elastogenesis is completed, this subunit is essential for extracellular signal transduction. Inefficiency of this receptor in old animals (30M) can be attributed to its uncoupling from its transduction pathway, as previously shown on human cells. Finally, the age-dependent variations of circulating elastin peptide concentration and elastin-laminin receptor responsiveness to elastin peptides are two independent parameters which could influence the vascular tension regulation.

Aging↗
PubMed1997

Inhibitory effect of type 1 collagen gel containing alpha-elastin on proliferation and migration of vascular smooth muscle and endothelial cells.

The purpose of this study was to investigate in vitro the potential effect of type 1 collagen gel containing alpha-elastin on the proliferation of vascular smooth muscle cells and vascular endothelial cells, and on smooth muscle cell migration. Vascular smooth muscle cell and endothelial cell were cultured in 12-well plates precoated with collagen gels and alpha-elastin. Cell proliferation rates were measured by monitoring [3H]-thymidine incorporation. After 2, 3 or 4 days of culture, the proliferation rate of both smooth muscle cells and endothelial cells was significantly decreased on collagen gel containing 10 mg/ml alpha-elastin compared with collagen gel only as control. Smooth muscle cell proliferation on collagen gel containing alpha-elastin on the 4th day of culture was decreased dose-dependently, e.g. 1 mg/ml of alpha-elastin (74.8(2.3)% of control, P=n.s.); 5 mg/ml (56.7(2.1)%; P<0.05); 10 mg/ml (30.3(3.1)%; P<0.005). In the case of cultured endothelial cells, however, [3H]-thymidine incorporation was not decreased significantly in the presence of 5 mg/ml alpha-elastin (83.1(7.9)%, P=n.s.). After stimulation by platelet-derived growth factor, the smooth muscle cell migration rate on collagen gel containing alpha-elastin (5 mg/ml) was decreased over time. The area of migration on the 6th day of culture was also significantly decreased dose-dependently in the presence of alpha-elastin, e.g. 1 mg/ml (72.6(3.4)% of control, P<0.05), 5 mg/ml (56.9%(1.5)%; P<0.05); 10 mg/ml (37.3(2.7)%; P<0.0005). In conclusion, alpha-elastin inhibited the proliferation and migration of smooth muscle cell in a dose-dependent manner on collagen gel culture, however, at high concentrations of alpha-elastin (10 mg/ml), the endothelial cell proliferation rate was also inhibited. At 5 mg/ml, alpha-elastin significantly inhibited smooth muscle cell proliferation and migration but did not significantly inhibit endothelial cell proliferation. Incorporation of collagen gel containing alpha-elastin into the structure of arterial prosthesis offers the possibility of inhibiting smooth muscle cell hyperplasia without significant effect on endothelial cell formation.

Animals↗
PubMed2005

A stereoelectronic effect on turn formation due to proline substitution in elastin-mimetic polypeptides.

Stereoelectronic effects have been identified as contributing factors to the conformational stability of collagen-mimetic peptide sequences. To assess the relevance of these factors within other protein structural contexts, three polypeptide sequences were prepared in which the sequences were derived from the canonical repeat unit (Val-Pro-Gly-Val-Gly) of the protein material elastin. These elastin-mimetic polypeptides, elastin-1, elastin-2, and elastin-3, incorporate (2S)-proline, (2S,4S)-4-fluoroproline, and (2S,4R)-4-fluoroproline, respectively, at the second position of the elastin repeat. Calorimetric and spectroscopic investigations of these three polypeptides indicate that the incorporation of the substituted proline residues had a dramatic effect upon the self-assembly of the corresponding elastin peptide. The presence of (2S,4R)-4-fluoroproline in elastin-3 lowered the temperature of the phase transition and increased the type II beta-turn population with respect to the parent polypeptide, while the presence of (2S,4S)-4-fluoroproline in elastin-2 had the opposite effect. These results suggest that stereoelectronic effects could either enhance or hinder the self-assembly of elastin-mimetic polypeptides, depending on the influence of the proline analogue on the energetics of the beta-turn conformation that develops within the pentapeptide structural repeats above the phase transition. Density functional theory (DFT) was employed to model three possible turn types (betaI-, betaII-, and inverse gamma-turns) derived from model peptide segments (MeCO-Xaa-Gly-NHMe) (Xaa = Pro, 4S-F-Pro, or 4R-F-Pro) corresponding to the turn-forming residues of the elastin repeat unit (Val-Pro-Gly-Val-Gly). The results of the these calculations suggested a similar outcome to the experimental data for the elastin-mimetic polypeptides, in that type II beta-turn structures were stabilized for peptide segments containing (2S,4R)-fluoroproline and destabilized for segments containing (2S,4S)-fluoroproline relative to the canonical proline residue.

Biomimetic Materials↗
PubMed1986

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.

Animals↗