The mineralization process of insoluble elastin fibrillar structures: Ionic environment vs degradation.

Despite its long half-life and physiological role, elastin undergoes irreversible changes (i.e elastolysis and/or calcification) impairing resilience of soft connective tissues. At present, it is still undefined: 1) to which extent elastin fibers have to be fragmented in order to increase their susceptibility to calcify; 2) which is the contribution of ionic environment on elastin mineralization; 3) why, in the same tissue area, mineralized coexist with non-mineralized fibers. The in vitro mineralization process was investigated on insoluble elastin, hydrolyzed or not-hydrolyzed, and incubated in different cell-free ionic environments. Mineral deposition is favored on hydrolyzed fibrillar structures due to exposure of multiple charged sites increasing the adsorption of Ca2+ that can attract phosphate and increase the local ion concentration over the point of supersaturation, representing the minimum requirement for hydroxyapatite nucleation sites. At physiological pH, the degree of elastin mineralization is influenced by hydrolysis and complexity of medium composition, since ionic species, as sodium, potassium, magnesium, in addition to calcium and phosphorus, interfere with the calcification process. These findings broaden the knowledge on the factors controlling hydroxyapatite deposition on insoluble elastin and can also explain why, in vivo, calcified and non-calcified fibers can be observed within the same tissue.

Exome sequencing and bioinformatic approaches reveals rare sequence variants involved in cell signalling and elastic fibre homeostasis: new evidence in the development of ectopic calcification.

Elastic fibres undergo aberrant mineralization in genetic as well as in acquired pathologic conditions causing severe impairment of tissue mechanical properties. Despite the number of investigations performed so far, the pathogenesis of these alterations is still elusive, due to both the complexity of the elastin network and the involvement of many genes and/or pro-osteogenic signalling pathways. Whole Exome Sequencing (WES) was performed on DNA from three patients affected by beta-thalassemia exhibiting soft connective tissue calcification. WES data were analysed with a bioinformatic approach, allowing to screen and to select genes carrying rare sequence variants. These genes were matched with those present in Extracellular Matrix DB. This approach enables to shed light on the involvement of the extracellular matrix in the occurrence of ectopic calcification. Results revealed a number of rare sequence variants in genes related to elastic fibre assembly and integrity. For instance, the involvement of fibrillins and collagen type VI in the formation of a modified microfibrillar scaffold may lead to elastic fibres less resilient and more prone to hydroxyapatite deposition. Moreover, data reveal that changes in mitochondrial metabolic pathways are sustained by a genetic background and emphasize that a persistent chronic oxidative stress can further influence extracellular matrix homeostasis and cell signalling through the TGFß-BMP axis. Eventually, the presence of multiple rare sequence variants in the Solute Carrier Family 25 Member 5 (SLC25A5) gene is suggestive of the role of this gene as a key factor linking mitochondria metabolism, ADP/ATP ratio and oxidative stress thus affecting extracellular matrix homeostasis and activation of pro-osteogenic factors.

Rare Co-occurrence of Beta-Thalassemia and Pseudoxanthoma elasticum: Novel Biomolecular Findings.

A number of beta-thalassemia patients, independently from the type of beta-thalassemia (ß0 or ß+) and blood transfusion requirements, may develop, after puberty, dermal, cardiovascular, and ocular complications associated with an ectopic mineralization phenotype similar to that observed in another rare genetic disorder, namely, Pseudoxanthoma elasticum (PXE). To date, the causes of these alterations in beta-thalassemia patients are not known, but it has been suggested that they could be the consequence of oxidative stress-driven epigenetic regulatory mechanisms producing an ABCC6 down-regulation. Since, in the last years, several genes have been associated to the ectopic mineralization phenotype, this study, for the first time, applied, on beta-thalassemia patients with ectopic mineralization phenotype, a multigene testing strategy. Selection of genes to be analyzed was done on the basis of (i) their genetic involvement in calcification diseases or (ii) their role in calcium-phosphate equilibrium. Although, due to the rarity of these conditions, a limited number of patients was analyzed, the detection of pathogenic variants represents the proof of concept that PXE and beta-thalassemia traits co-occur on a genetic basis and that, in addition to causative mutations, functional polymorphisms may further influence connective tissue manifestations. The use of a multigene-based next-generation sequencing represents a useful time- and cost-effective approach, allowing to identify sequence variants that might improve prognostic assessment and better management of these patients, especially in the current era of precision medicine aiming to identify individual optimal care based on a unique personal profile.

Interactions between elastin-like peptides and an insulating poly(ortho-aminophenol) membrane investigated by AFM and XPS.

This investigation was undertaken to explore the mutual recognition of the pentapeptide (ValGlyGlyValGly)n, a hydrophobic elastin-like peptide (ELP), suspended in deionized water in monomer (n = 1) and trimer (n = 3) forms and the outer surface of a very thin, insulating polymer, poly(ortho-aminophenol) (PoAP), electrochemically grown on a platinum foil by cyclic voltammetry in a neutral medium (phosphate-buffered saline, I = 0.1M) immersed in the suspension. As a prior task, the proved propensity of the ValGlyGlyValGly sequence, at the given minimal length (three or more repeats), to self-assemble into amyloid-like fibrils when solubilized in an aqueous environment was considered within the framework of testing PoAP surfaces for the specific detection of amyloid precursors. From our knowledge of the chemical structure and physical properties of both biomacromolecule families obtained in previous studies, we focused on the efficacy of the binding sites offered to ELP fibrils by PoAP in its as-prepared form or properly modified either by postsynthesis oxidation or by adsorption/entrapping of ELP monomer(s) with or without protecting terminal groups. Consistent with all methods of preparation, the best surfaces, recognizable by the trimer fibrils, are those modified to carry a larger number of carbonyls, particularly by entrapment of ELP monomer(s) during PoAP electrosynthesis using an imprinting-inspired method. The degree of attachment of fibrillar aggregates, detected by atomic force microscopy and X-ray photoelectron spectroscopy, provides unequivocal evidence of the cooperative forces involving PoAP-ELP interactions. The results obtained suggest the prospect of using the proposed Pt/PoAP/ELP systems as biodetectors in Alzheimer disease. Graphical abstract Synthesis steps of Pt/PoAP/ELP electrodes for amyloid detection. AFM = Atomic Force Microscopy, CV = Cyclic Voltammetry, ELPs = Elastin like Peptides, PoAP = Poly ortho-Aminophenol, Pt = Platinum, XPS = X-ray Photoelectron Spectroscopy.

Heparan sulfates facilitate harmless amyloidogenic fibril formation interacting with elastin-like peptides.

Heparan sulfates (HSs) modulate tissue elasticity in physiopathological conditions by interacting with various matrix constituents as tropoelastin and elastin-derived peptides. HSs bind also to protein moieties accelerating amyloid formation and influencing cytotoxic properties of insoluble fibrils. Interestingly, amyloidogenic polypeptides, despite their supposed pathogenic role, have been recently explored as promising bio-nanomaterials due to their unique and interesting properties. Therefore, we investigated the interactions of HSs, obtained from different sources and exhibiting various degree of sulfation, with synthetic amyloidogenic elastin-like peptides (ELPs), also looking at the effects of these interactions on cell viability and cell behavior using in vitro cultured fibroblasts, as a prototype of mesenchymal cells known to modulate the soft connective tissue environment. Results demonstrate, for the first time, that HSs, with differences depending on their sulfation pattern and chain length, interact with ELPs accelerating aggregation kinetics and amyloid-like fibril formation as well as self-association. Furthermore, these fibrils do not negatively affect fibroblasts' cell growth and parameters of redox balance, and influence cellular adhesion properties. Data provide information for a better understanding of the interactions altering the elastic component in aging and in pathologic conditions and may pave the way for the development of composite matrix-based biomaterials.

Structural characterization and biological properties of the amyloidogenic elastin-like peptide (VGGVG)3.

The peculiar and unique properties of elastin are due to the abundance of hydrophobic residues and of repetitive sequences as XGGZG (X, Z=V, L or A). Unexpectedly, these sequences not only provide elasticity to the whole protein, but are also able to form amyloid-like fibrils. Even though amyloid fibrils have been associated for a long time to the development of serious disorders as Alzheimer's disease, recent evidence suggests that toxicity may be related to oligomeric species or to pre-fibrillar intermediates, rather than to mature fibrils. In addition, a number of studies highlighted the potential of "bio-inspired" materials based on amyloid-like nanostructures. The present study has been undertaken with the aim to characterize a chemically synthesized elastin-like peptide (VGGVG)3. Structural and biological features were compared with those of peptides as poly(VGGVG) and VGGVG that, having the same amino acid sequence, but different length and supramolecular structure have been previously investigated for their amyloidogenic properties. Results demonstrate that a minimum sequence of 15 amino acids is sufficient to aggregate into short amyloid-like fibrils, whose formation is however strictly dependent on the specific VGGVG repeated sequence. Moreover, in the attempt to elucidate the relationship among aggregation properties, fibers morphology and biocompatibility, 3T3 fibroblasts were grown in the presence of VGGVG-containing elastin-like peptides (ELPs) and analyzed for their ability to proliferate, attach and spread on ELPs-coated surfaces. Data clearly show that amyloid-like fibrils made of (VGGVG)3 are not cytotoxic at least up to the concentration of 100 µg/ml, even after several days of culture, and are a good support for cell attachment and spreading.

Combined effects of solvation and aggregation propensity on the final supramolecular structures adopted by hydrophobic, glycine-rich, elastin-like polypeptides.

Previous work on elastin-like polypeptides (ELPs) made of hydrophobic amino acids of the type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu) has consistently shown that differing dominant supramolecular structures were formed when the suspending media were varied: helical, amyloid-like fibers when suspended in water and globules evolving into "string of bead" structures, poly(ValGlyGlyValGly), or cigar-like bundles, poly(ValGlyGlyLeuGly), when suspended in methyl alcohol. Comparative experiments with poly(LeuGlyGlyValGly) have further indicated that the interface energy plays a significant role and that solvation effects act in concomitance with the intrinsic aggregation propensity of the repeat sequence. Continuing our investigation on ELPs using surface (X-ray photoelectron spectroscopy, atomic force microscopy) and bulk (circular dichroism, Fourier transform infrared spectroscopy) techniques for their characterization, here we have compared the effect of suspending solvents (H(2)O, dimethylsulfoxide, ethylene glycol, and MeOH) on poly(ValGlyGlyValGly), the polypeptide most inclined to form long and well-refined helical fibers in water, searching for the signature of intermolecular interactions occurring between the polypeptide chains in the given suspension. The influence of sequence specificities has been studied by comparing poly(ValGlyGlyValGly) and poly(LeuGlyGlyValGly) with a similar degree of polymerization. Deposits on substrates of the polypeptides were characterized taking into account the differing evaporation rate of solvents, and tests on their stability in ultra high vacuum were performed. Finally, combining experimental and computational studies, we have revaluated the three-dimensional modeling previously proposed for the supramolecular assembly in water of poly(ValGlyGlyValGly). The results were discussed and rationalized also in the light of published data.

Heparan sulfate affects elastin deposition in fibroblasts cultured from donors of different ages.

Heparan sulfate (HS), due to its presence on the cell surface and in the extracellular milieu and its ability to modulate cell signaling, has a fundamental role in both physiological and pathological conditions. For decades we have demonstrated the occurrence of interactions between glycosaminoglycans (GAGs) and elastic fibers. In particular, we have recently shown that HS is present inside elastic fibers and plays a role in the assembly and stability of elastin coacervates. Elastin represents, within the extracellular matrix, the component most severely affected during aging, and changes in the synthesis and posttranslational modifications of HS have been described, possibly influencing cellular behavior and protein interactions. Thus, the present study has investigated, in two different in vitro experimental models, the role of HS on elastin deposition and assembly. Results demonstrate that: (1) Biological effects of HS are partly dependent on the physicochemical characteristics of the GAGs; (2) HS does not affect attachment, viability, and growth of human dermal fibroblasts; (3) HS does not modify elastin gene expression nor elastin synthesis, but favors α-elastin aggregation and, independently from the age of donors, elastin assembly; (4) HS significantly increases the expression of fibulin 5, and these effects are especially evident in fibroblasts isolated from aging donors. These data provide a better understanding of the biological role of HS and offer new perspectives regarding the possibility of restoring and/or preserving the elastic component with aging.

Influence of amino acid specificities on the molecular and supramolecular organization of glycine-rich elastin-like polypeptides in water.

Elastin-like polypeptides adopt complex supramolecular structures, showing either a hydrophobic or a hydrophilic surface, depending on their surrounding environment and the supporting substrate. The preferred organization is important in many situations ranging from biocompatibility to bio-function. Here we compare the n-repeat pentamer LeuGlyGlyValGly (n = 7) with the analogue ValGlyGlyValGly (n = 5), as water suspensions and as deposits on silicon substrates. These sequences contain the repeat XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu) motif belonging to the hydrophobic glycine-rich domain of elastin and represent a simplified model from which to obtain information on molecular interactions functional to elastin itself. The compounds studied differ only by the presence of the -CH(2)- spacer in the Leu moiety and thus the work was aimed at revealing the influence of this spacer element on self assembly. Both polypeptides were studied under identical conditions, using combined techniques, to identify differences in their conformational states both at molecular (CD, FTIR) and supramolecular (XPS, AFM) levels. By these means, together with a Congo Red spectroscopic assay of ß-sheet formation in water, a clear correlation between amino acid sequences (sequence specificity) and their kinetics and ordering of aggregation has emerged. The novel outcomes of this work are from the supplementary measurements, made to augment the AFM and XPS studies, showing that the significant step in the self assembly of both polypeptides takes place in the liquid phase and from the finding that the substitution of Val by Leu in the first position of the pentapeptide effectively inhibits the formation of amyloidal fibers.