Relevance of aggregation properties of tropoelastin to the assembly and structure of elastic fibers

Bressan, Giorgio M.; Pasquali‐Ronchetti, I.; Fornieri, C.; Mattioli, F.; Castellani, I.; Volpin, Dino

doi:10.1016/0889-1605(86)90068-6

Cited by 82 publications

(87 citation statements)

References 16 publications

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“…36,37 Because the physico-chemical and biological properties of HA are often dependent on its size, 38 we have sought to investigate these differences in the context of stimulating elastin matrix synthesis. 16,17 In other studies, copper ions (Cu 2þ ) have been shown to minimize vascular defects, 39 promote angiogenesis, 40 and enhance LOX enzyme activity.…”

Section: Discussionmentioning

confidence: 99%

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Kothapalli

Ramamurthi

2009

Tissue Engineering Part A

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Current efforts to tissue engineer elastin-rich vascular constructs and grafts are limited because of the poor elastogenesis of adult vascular smooth muscle cells (SMCs) and the unavailability of appropriate cues to upregulate and enhance cross-linking of elastin precursors (tropoelastin) into organized, mature elastin fibers. We earlier showed that hyaluronan (HA) fragments greatly enhance tropo-and matrix-elastin synthesis by SMCs, although the yield of matrix elastin is low. To improve matrix yields, here we investigate the benefits of adding copper (Cu 2+ ) ions (0.01 M and 0.1 M), concurrent with HA (756-2000 kDa), to enhance lysyl oxidase (LOX)-mediated elastin cross-linking machinery. Although absolute elastin amounts in test groups were not different from those in controls, on a per-cell basis, 0.1 M of Cu 2+ ions slowed cell proliferation (5.6 AE 2.3-fold increase over 21 days vs 22.9 AE 4.2-fold for non-additive controls), stimulated synthesis of collagen (4.1 AE 0.4-fold), tropoelastin (4.1 AE 0.05-fold) and cross-linked matrix elastin (4.2 ± 0.7-fold). LOX protein synthesis increased 2.5 times in the presence of 0.1 M of Cu 2+ ions, and these trends were maintained even in the presence of HA fragments, although LOX functional activity remained unchanged in all cases. The abundance of elastin and LOX in cell layers cultured with 0.1 M of Cu 2+ ions and HA fragments was qualitatively confirmed using immunoflourescence. Scanning electron microscopy images showed that SMC cultures supplemented with 0.1 M of Cu 2+ ions and HA oligomers and large fragments exhibited better deposition of mature elastic fibers (*1 mm diameter). However, 0.01 M of Cu 2+ ions did not have any beneficial effect on elastin regeneration. In conclusion, the results suggest that supplying 0.1 M of Cu 2+ ions to SMCs to concurrently (a) enhance per-cell yield of elastin matrix while allowing cells to remain viable and synthetic and not density-arrested in long-term culture because of their moderating effects on otherwise rapid cell proliferation and (b) provide additional benefits of enhanced elastin fiber formation and cross-linking within these tissue-engineered constructs.

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Section: Discussionmentioning

confidence: 99%

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Kothapalli

Ramamurthi

2009

Tissue Engineering Part A

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“…Through coacervation, tropoelastin molecules were thought to be converted from molecules largely lacking secondary and tertiary structure to a more ordered state (2,7,9,10). Electron micrographs show parallel arrays of 5-nm-wide filaments of tropoelastin coacervates that are similar to the fibrous structure of mature elastin (3,4,11). The importance of coacervation in the formation of lysine cross-links has been shown in smooth muscle cells where culturing at temperatures below 37°C hampers elastin formation (12,13).…”

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confidence: 99%

“…Alternating between hydrophobic domains in the protein are short sequences of amino acid residues that form the cross-linking domains (6). Coacervation may concentrate and align tropoelastin molecules prior to elastin formation via lysyl oxidase-mediated cross-linkage of the lysine residues that leads to a growing elastic fiber (4,7,8).Vrhovski et al(1) demonstrated that maximal coacervation of recombinant human tropoelastin occurs under the physiologically relevant conditions of 37°C, 150 mM NaCl, and pH 7-8. Through coacervation, tropoelastin molecules were thought to be converted from molecules largely lacking secondary and tertiary structure to a more ordered state (2, 7, 9, 10).…”

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confidence: 99%

“…Tropoelastin is soluble in aqueous solution at room temperature in vitro, but upon raising the temperature to 37°C the solution becomes turbid as tropoelastin molecules associate to form large aggregates (2,3). This process of coacervation results from multiple intermolecular interactions of the hydrophobic domains (3,4). The tropoelastin coacervate is a thick viscoelastic phase that is not miscible with the overlying solution (5).…”

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Thermodynamic and Hydrodynamic Properties of Human Tropoelastin

Toonkool¹,

Regan²,

Kuchel³

et al. 2001

Journal of Biological Chemistry

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Tropoelastin is the soluble precursor of elastin that bestows tissue elasticity in vertebrates. Tropoelastin is soluble at 20°C in phosphate-buffered saline, pH 7.4, but at 37°C equilibrium is established between soluble protein and insoluble coacervate. Sedimentation equilibrium studies performed before (20°C) and after (37°C) coacervation showed that the soluble component was strictly monomeric. Sedimentation velocity experiments revealed that at both temperatures soluble tropoelastin exists as two independently sedimenting monomeric species present in approximately equal concentrations. Species 1 had a frictional ratio at both temperatures of ϳ2.2, suggesting a very highly expanded or asymmetric protein. Species 2 displayed a frictional ratio at 20°C of 1.4 that increased to 1.7 at 37°C, indicating a compact and symmetrical conformation that expanded or became asymmetric at the higher temperature. The slow interconversion of the two monomeric species contrasts with the rapid and reversible process of coacervation suggesting both efficiently incorporate into the coacervate. A hydrated protein of equivalent molecular weight modeled as a sphere and a flexible chain was predicted to have a frictional ratio of 1.2 and 1.6, respectively. Tropoelastin appeared as a single species when studied by pulsed field-gradient spinecho NMR, but computer modeling showed that the method was insensitive to the presence of two species of equal concentration having similar diffusion coefficients. Scintillation proximity assays using radiolabeled tropoelastin and sedimentation analysis showed that the coacervation at 37°C was a highly cooperative monomer-n-mer self-association. A critical concentration of 3.4 g/liter was obtained when the coacervate was modeled as a helical polymer formed from the monomers via oligomeric intermediates.Elastin forms a highly insoluble cross-linked extracellular matrix that is predominantly responsible for the elasticity of vertebrate tissue. The precursor of elastin, tropoelastin, is devoid of cross-links. Following secretion from the cell surface, tropoelastin undergoes coacervation, which is a process of selfassociation characterized by an inverse temperature transition (1). Tropoelastin is soluble in aqueous solution at room temperature in vitro, but upon raising the temperature to 37°C the solution becomes turbid as tropoelastin molecules associate to form large aggregates (2, 3). This process of coacervation results from multiple intermolecular interactions of the hydrophobic domains (3, 4). The tropoelastin coacervate is a thick viscoelastic phase that is not miscible with the overlying solution (5). On cooling to 20°C, the aggregates dissociate reversibly, and the solution turns clear. Alternating between hydrophobic domains in the protein are short sequences of amino acid residues that form the cross-linking domains (6). Coacervation may concentrate and align tropoelastin molecules prior to elastin formation via lysyl oxidase-mediated cross-linkage of the lysine residues that leads to a grow...

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Chemical synthesis of cross-linked poly(KGGVG), an elastin-like biopolymer

Martino

Tamburro

2001

Biopolymers

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Previous studies afforded on peptides and polypeptides containing repetitive sequences of elastin have largely demonstrated that their molecular and supramolecular properties are fully representative of those of tropoelastin, the soluble, linear precursor of elastin itself. In the attempt to synthesize cross‐linked elastin‐mimetic polypeptides, the repeating sequence VGGVG (V: valine; G: glycine), typical of elastin, was modified to incorporate lysine residues, yielding the polymer poly(KGGVG) (K: lysine). This imparts primary amine functionality susceptible to cross‐linking reaction with appropriate bifunctional cross‐linking reagents. We report herein the chemical synthesis and cross‐linking of poly(KGGVG) with glutaraldehyde (GTA) and with disuccinimidyl glutarate (DSG). In both cases, the characterization of the polymers, both linear and cross‐linked, has been carried out by CD spectroscopy and transmission electron microscopy measurements. The obtained results, although not conclusive, demonstrate that poly(KGGVG), both linear and cross‐linked, may be considered very similar to tropoelastin and mature elastin, as concerns its molecular and supramolecular properties. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 29–37, 2001

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Relevance of aggregation properties of tropoelastin to the assembly and structure of elastic fibers

Cited by 82 publications

References 16 publications

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Thermodynamic and Hydrodynamic Properties of Human Tropoelastin

Chemical synthesis of cross-linked poly(KGGVG), an elastin-like biopolymer

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