Changes in the blood vessel wall elastin of spontaneously hypertensive (SHR) rats

It has been demonstrated that both elastin and tropoelastin preparations obtained from aortae of spontaneously hypertensive rats at the stage of established hypertension differ in their amino acid composition from age-matching controls. The differences refer to an increased proportion of polar amino acids, particularly aspartic and glutamic acid (about a two-fold increase compared to the controls) and arginine and tyrosine (1.5 times the control value). On the other hand, this increase is compensated for by a decrease in the valine concentration. Furthermore, direct estimation of the number of val-pro sequence in different elastin preparations indicated a drop from 49.3 to 29.2 per 1,000 residues in normotensive controls and preparations obtained from spontaneously hypertensive rats respectively.     

Variations in aortal elastin composition after atherogenic diet feeding in rats. Possible involvement of two different elastin types

An increased proportion (about twofold) of polar amino acids (aspartic acid, glutamic acid, arginine and tyrosine) was found both in insoluble elastin and tropoelastin preparations from aortae of rats fed high fat diet. Concomitantly the number of val-pro sequences drops from 48.7 to 26.8 in elastin from control and atheromatous aortae. The observed changes can be explained either by assuming a tight attachment of a non-elastin protein rich in polar amino acids or by assuming the existence two elastin types the proportion of which is changed in aortae by high fat diet feeding. The data obtained are in favor of two different genetic types of elastin.     

Carbohydrate content of insoluble elastins prepared from adult bovine and calf ligamentum nuchae and tropoelastin isolated from copper-deficient procine aorta

1. Insoluble elastin has been prepared by several different methods from adult bovine and calf ligamentum nuchae. Highly purified tropoelastin has been prepared from copper-deficient porcine aorta. 2. Amino acid analyses indicated that all preparations, except that obtained from calf ligamentum nuchae by using an EDTA extraction followed by collagenase digestion (preparation E6), were typical of pure elastin having high concentrations of hydrophobic and low concentrations of hydrophilic amino acids. Preparation E6 was found to contain approx. 40% collagen. 3. The determination and composition of the carbohydrates associated with these preparations is reported. With the exception of preparation E6, the insoluble elastins contained only trace amounts of neutral sugars (0.13-0.35%, w/w) and amino sugars (0.01-0.06%, w/w). The porcine tropoelastin contained virtually no carbohydrate. 4. The results suggest that carbohydrate analyses can yield valuable information about the purity of elastin preparations.     

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.     

Binding of elastin to Staphylococcus aureus.

Many pathogenic bacteria specifically bind to components of the extracellular matrix. In this study, we report the specific association of Staphylococcus aureus with elastin, a major structural component of elastic tissue. Competition assays in which the binding of radiolabeled tropoelastin was inhibited by excess unlabeled elastin peptides, but not by other proteins, established the specificity of the interaction. Kinetic studies showed that tropoelastin binding to the bacteria was rapid and saturable. Scatchard analysis of the equilibrium binding data indicated the presence of a single class of high affinity binding sites (KD approximately 4-7 nM) with approximately 1000 sites per organism. Protease susceptibility suggested that the elastin binding moiety on S. aureus was a protein, which was confirmed by the isolation of a 25-kDa elastin-binding protein from S. aureus extracts through affinity chromatography. Using a truncated form of tropoelastin, the bacterial binding domain on elastin was mapped to a 30-kDa fragment at the amino end of the molecule. Although the precise amino acid sequence recognized by the staphylococcal elastin receptor has not been characterized, it is clearly different from the region of tropoelastin that specifies binding to mammalian elastin receptors.     

Extracellular matrix: differential influence on growth and biosynthesis patterns of vascular smooth muscle cells from SHR and WKY rats

Smooth muscle cells from spontaneously hypertensive rats (SHR) elaborated extracellular matrix (ECM) material in culture that was more stimulatory to growth of cells from normotensive (WKY) animals than their own matrix. Both cell types elaborated ECMs consisting of glycoconjugate material (proteoglycans, glycopeptides) elastin, and collagens, but there were differences in the relative proportions of the compounds synthesized. Cells from SHR produced an ECM richer in elastin than that synthesized by WKY derived cells (approximately 19% vs. 11%, respectively). However, the latter elaborated ECMs containing more (approximately 45% for WKY vs. 29% for SHR) glycoconjugate material than the former. The lysyloxidase-mediated cross-linking of elastin was more rapid in cultured cells from SHR animals than from their normotensive counterparts and may be as a consequence of increased substate (tropoelastin) availability in ECMs from SHR animals. The relative proportions and sulphate levels of the glycosaminoglycans associated with matrix material elaborated by the two cell types were similar. Radiolabelled glycoconjugate material was degraded by cells (SHR/WKY) when they were plated upon pre-formed ECMs, and their patterns of synthesis of new matrix was markedly altered under such conditions. New matrix material elaborated by cells plated upon ECM-coated dishes consisted predominantly of glycopeptide and proteoglycan matrix components. Epidermal growth factor promoted the incorporation of [3H]-thymidine into DNA by quiescent cells, and this was also markedly stimulated when cells were plated onto ECM-coated plasticware rather than onto plastic substratum.     

The action of neutrophil serine proteases on elastin and its precursor

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P₁, which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P₁ in tropoelastin, whereas Lys was not identified at P₁ in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P₂ and P₄′. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG.     

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).     

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.     

Partial characterization of a tropoelastin precursor isolated from chick aorta.

Evidence is presented that indicates tropoelastin is derived from a soluble elastin with a molecular weight of 95000. Tropoelastin and its proposed precursor were isolated from the aortas of copper-deficient chicks. Although it is doubtful that the proposed precursor is an initial product of elastin translation, i.e., a proelastin, it is proposed to be at least a truncated form of proelastin that is converted to tropoelastin. The key to its isolation was the presence of alpha 1-antitrypsin at each step in the purification procedure. The first 11 amino acid residues at the NH2 terminal of the proposed tropoelastin precursor (GGVPGVAVPGGV) are the same as those for tropoelastin. Its amino acid composition is similar to that of tropoelastin, except for higher amounts of acidic amino acid residues. Further, the proposed precursor contains a limited number of aldehydic functions, presumably in the form of peptidyl allysine. This was taken as an indication that the proposed precursor serves as a substract for lysyl oxidase. Under the conditions used for the isolation, the precursor appeared to be in higher concentrations than tropoelastin in aorta extracts from copper-deficient chicks.     

Effect of α-Amino-4-Phosphonobutyrate on the Release of Endogenous Glutamate and Aspartate from Cortical Synaptosomes of Epileptic Rats

The effect of the glutamate antagonist alpha-amino-4-phosphonobutyrate (APBA) on the release of endogenous amino acids from sensorimotor cortical synaptosomes of rats with a cortical cobalt focus and from non-epileptic rats was studied: (1) The release of endogenous glutamate, aspartate, and gamma-aminobutyric acid (GABA) from synaptosomal preparations of cobalt-induced epileptogenic tissues was increased compared with the release from the contralateral (sensorimotor) region or the sensorimotor cortex of normal animals. The intrasynaptosomal content of these amino acids was reduced in proportion to the amount released. The levels of other amino acids were unaffected or showed much smaller changes. (2) APBA (0.5-1 mM) decreased significantly the spontaneous release of aspartate and glutamate from the epileptic foci without affecting GABA or any other amino acid. (3) APBA produced no effect whatsoever on the release of any amino acid from synaptosomal preparations of nonepileptic focus.     

Isolation of tropoelastin a from lathyritic chick aortae.

Tropoelastin a was isolated from lathyritic chick aortae by using severe denaturing conditions for the initial extraction. The amino acid composition of this new species of tropoelastin is elastin-like in its high proportion of proline, glycine, alanine and valine. However, it differs from authentic tropoelastin b in containing a higher percentage of polar amino acids and cysteine residues. In addition, the amount of proline hydroxylation is 3 times higher than that found in chick tropoelastin b.     

Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.     

Amino acid sequences C-terminal to the cross-links in bovine elastin.

All the desmosine-containing elastolytic peptides of bovine ligamentum-nuchae elastin have now been examined for amino acid sequences C-terminal to the cross-links. In addition, amino acid residues C-terminal to lysine residues in bovine tropoelastin were also examined. No tyrosine C-terminal to cross-links in bovine elastin or C-terminal to lysine in tropoelastin was detected. Apparently all the tyrosine residues C-terminal to lysine residues in pig tropoelastin are replaced with phenylalanine in bovine tropoelastin. All the data presented are consistent with the scheme proposed for the formation of desmosine and isodesmosine cross-links of elastin by Gerber & Anwar [(1975) Biochem. J. 149, 685--695].     

Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells

Neonatal rat aortic smooth muscle cell cultures produce two major soluble elastin molecules termed protropoelastin (77 kDa) and tropoelastin (71 kDa). Cell layer extracts are protroproelastin-enriched, while protropoelastin, tropoelastin, and significant amounts of discrete elastin fragments (Mr of 66,000, 61,000, 56,000, and 45,000) are present in preparations from the medium of these cultures. To determine the role of the various elastin molecules in the metabolism of elastin in neonatal rat aortic smooth muscle cell cultures, the amino termini of these proteins were sequenced. All soluble elastin components present in the medium were purified as a single peak by high performance liquid chromatography; further separation of the components was achieved by polyacrylamide gel electrophoresis and electroblotting. The bands were excised and sequenced. The amino-terminal sequences of protropoelastin, tropoelastin, and the 66-kDa, 61-kDa, and 56-kDa fragments were identical: Gly-Gly-Val-Pro-Gly-Ala-Val-Pro-Gly-Gly. This sequence is identical with published amino-terminal sequences of tropoelastins from several other species. As expected, when the cell cultures were pulsed with [3H]valine, all the soluble elastin molecules were radioactive, while only protropoelastin appeared radioactive after [35S] cysteine pulsing. Since cysteine is present only in the carboxyl-terminal end of the molecule, all the data indicate that the cleavage of the elastin fragments identified in the culture are occurring at the carboxyl end of protropoelastin. These results are consistent with the original hypothesis that a precursor-product relationship exists between the 77-kDa and 71-kDa soluble elastin molecules. Based on known tropoelastin sequences and the molecular weights of the discrete fragments, additional fragmentation of protropoelastin and/or tropoelastin most likely occurs at the lysine/alanine-enriched domains presumably involved in cross-link formation.     

Effect of starvation and refeeding on amino acid uptake by mammary gland of the lactating rat. Role of ketone bodies.

Arteriovenous differences of amino acids across the mammary glands of lactating rats are diminished when the rats are starved for 24 h. When 24 h-starved rats were refed for 2 1/2 h, the arteriovenous differences of amino acids returned to values similar to those found in well-fed rats. In order to find a possible explanation for these rapid changes, we tested the effect of ketone bodies on amino acid uptake by the gland. At 5 min after injection of acetoacetate to fed rats, when the total concentration of ketone bodies in blood was similar to that found in starvation, the uptake of amino acids by the mammary gland was similar to that found after starvation, i.e. lower than in fed rats. However, 30 min after administration of acetoacetate, when the arterial concentration of ketone bodies had returned to values similar to those in fed rats, the arteriovenous differences of amino acids were similar to those found in fed rats. We conclude that the changes in blood ketone bodies may be responsible, at least in part, for the changes in amino acid uptake that occur in starvation and in the starvation--refeeding transition.     

Heightened aberrant deposition of hard-wearing elastin in conduit arteries of prehypertensive SHR is associated with increased stiffness and inward remodeling

Elastin is a major component of conduit arteries and a key determinant of vascular viscoelastic properties. Aberrant organization of elastic lamellae has been reported in resistance vessels from spontaneously hypertensive rats (SHR) before the development of hypertension. Hence, we have characterized the content and organization of elastic lamellae in conduit vessels of neonatal SHR in detail, comparing the carotid arteries from 1-wk-old SHR with those from Wistar-Kyoto (WKY) and Sprague Dawley (SD) rats. The general structure and mechanics were studied by pressure myography, and the internal elastic lamina organization was determined by confocal microscopy. Cyanide bromide-insoluble elastin scaffolds were also prepared from 1-mo-old SHR and WKY aortas to assess their weight, amino acid composition, three-dimensional lamellar organization, and mechanical characteristics. Carotid arteries from 1-wk-old SHR exhibited narrower lumen and greater intrinsic stiffness than those from their WKY and SD counterparts. These aberrations were associated with heightened elastin content and with a striking reduction in the size of the fenestrae present in the elastic lamellae. The elastin scaffolds isolated from SHR aortas also exhibited increased relative weight and stiffness, as well as the presence of peculiar trabeculae inside the fenestra that reduced their size. We suggest that the excessive and aberrant elastin deposited in SHR vessels during perinatal development alters their mechanical properties. Such abnormalities are likely to compromise vessel expansion during a critical period of growth and, at later stages, they could compromise hemodynamic function and participate in the development of systemic hypertension.     

Methods in elastic tissue biology: elastin isolation and purification

Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60-70 kDa monomer called tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin 'contaminates' are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and tropoelastin. It also addresses key aspects of studying tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.     

Analysis of the 3' region of the sheep elastin gene

The nucleotide sequences of a 1279-bp sheep elastin cDNA clone, pcSEL1 [Yoon et al. (1984) Biochem. Biophys. Res. Commun. 118, 261-269], and a 1230-bp sheep elastin genomic subclone, pSS1 [Davidson et al. (1984) Biochem. J. 220, 643-652], corresponding to a portion of the cDNA clone, were determined. These analyses permitted determination of the 100 amino acids at the carboxy terminus of sheep tropoelastin. A portion of this sequence showed strong homology to known sequences of pig tropoelastin, but most of the sequence had not been previously determined through protein sequencing. Novel aspects of the tropoelastin molecule which have been revealed by the present analyses are (i) the presence of an unusual sequence, KPPKP, which may contribute to crosslink formation; and (ii) the finding of cysteine within a sequence, CLGKSCGRKRK, at the putative carboxy terminus of tropoelastin. Because of the presence of these sequences, it is speculated that the carboxy-terminal region may be of importance in crosslinking tropoelastin molecules to themselves or to other matrix macromolecules. The nucleotide analyses revealed that sheep elastin mRNA contains a 974-bp untranslated sequence at the 3' end, which appears to be strongly conserved among species.     

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

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