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Fibroblast to myofibroblast differentiation is a key feature of wound-healing in soft tissues, including the vagina. Vaginal fibroblasts maintain the integrity of the vaginal wall tissues, essential to keep pelvic organs in place and avoid pelvic organ prolapse (POP). The micro-environment of vaginal tissues in POP patients is stiffer and has different extracellular matrix (ECM) composition than healthy vaginal tissues. In this study, we employed a series of matrices with known stiffnesses, as well as vaginal ECMs, in combination with vaginal fibroblasts from POP and healthy tissues to investigate how matrix stiffness and composition regulate myofibroblast differentiation in vaginal fibroblasts. Stiffness was positively correlated to production of α-smooth muscle actin (α-SMA). Vaginal ECMs induced myofibroblast differentiation as both α-SMA and collagen gene expressions were increased. This differentiation was more pronounced in cells seeded on POP-ECMs that were stiffer than those derived from healthy tissues and had higher collagen and elastin protein content. We showed that stiffness and ECM content regulate vaginal myofibroblast differentiation. We provide preliminary evidence that vaginal fibroblasts might recognize POP-ECMs as scar tissues that need to be remodeled. This is fundamentally important for tissue repair, and provides a rational basis for POP disease modelling and therapeutic innovations in vaginal reconstruction.

Elastic fibers are essential assemblies of vertebrates and confer elasticity and resilience to various organs including blood vessels, lungs, skin, and ligaments. Mature fibers, which comprise a dense and insoluble elastin core and a microfibrillar mantle, are extremely resistant toward intrinsic and extrinsic influences and maintain elastic function over the human lifespan in healthy conditions. The oxidative deamination of peptidyl lysine to peptidyl allysine in elastin's precursor tropoelastin is a crucial posttranslational step in their formation. The modification is catalyzed by members of the family of lysyl oxidases and the starting point for subsequent manifold condensation reactions that eventually lead to the highly cross-linked elastomer. This review summarizes the current understanding of the formation of cross-links within and between the monomer molecules, the molecular sites, and cross-link types involved and the pathological consequences of abnormalities in the cross-linking process.

BACKGROUND: Skin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides.METHODS: Cathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry.RESULTS: Elastin samples derived from patients between 10 and 86 years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies.CONCLUSION: Cathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.

Lysyl oxidases (LOXs) play a central role in extracellular matrix remodeling during development and tumor growth and fibrosis through cross-linking of collagens and elastin. We have limited knowledge of the structure and substrate specificity of these secreted enzymes. LOXs share a conserved C-terminal catalytic domain but differ in their N-terminal region, which is composed of 4 repeats of scavenger receptor cysteine-rich (SRCR) domains in LOX-like (LOXL) 2. We investigated by X-ray scattering and electron microscopy the low-resolution structure of the full-length enzyme and the structure of a shorter form lacking the catalytic domain. Our data demonstrate that LOXL2 has a rod-like structure with a stalk composed of the SRCR domains and the catalytic domain at its tip. We detected direct interaction between LOXL2 and tropoelastin (TE) and also LOXL2-mediated deamination of TE. Using proteomics, we identified several allysines together with cross-linked TE peptides. The elastin-like material generated was resistant to trypsin proteolysis and displayed mechanical properties similar to mature elastin. Finally, we detected the codistribution of LOXL2 and elastin in the vascular wall. Altogether, these data suggest that LOXL2 could participate in elastogenesis in vivo and could be used as a means of cross-linking TE in vitro for biomimetic and cell-compatible tissue engineering purposes.-Schmelzer, C. E. H., Heinz, A., Troilo, H., Lockhart-Cairns, M.-P., Jowitt, T. A., Marchand, M. F., Bidault, L., Bignon, M., Hedtke, T., Barret, A., McConnell, J. C., Sherratt, M. J., Germain, S., Hulmes, D. J. S., Baldock, C., Muller, L. Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin.

In addition to its critical role in lysosomes for catabolism of sialoglycoconjugates, NEU1 is expressed at the plasma membrane and regulates a myriad of receptors by desialylation, playing a key role in many pathophysiological processes. Here, we developed a proteomic approach dedicated to the purification and identification by LC-MS/MS of plasma membrane NEU1 interaction partners in human macrophages. Already known interaction partners were identified as well as several new candidates such as the class B scavenger receptor CD36. Interaction between NEU1 and CD36 was confirmed by complementary approaches. We showed that elastin-derived peptides (EDP) desialylate CD36 and that this effect was blocked by the V14 peptide, which blocks the interaction between bioactive EDP and the elastin receptor complex (ERC). Importantly, EDP also increased the uptake of oxidized LDL by macrophages that is blocked by both the V14 peptide and the sialidase inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA). These results demonstrate, for the first time, that binding of EDP to the ERC indirectly modulates CD36 sialylation level and regulates oxidized LDL uptake through this sialidase. These effects could contribute to the previously reported proatherogenic role of EDP and add a new dimension in the regulation of biological processes through NEU1.

Matrix metalloproteinases are a class of enzymes, which degrade extracellular matrix components such as collagens, elastin, laminin or fibronectin. So far, four matrix metalloproteinases have been shown to degrade elastin and its precursor tropoelastin, namely matrix metalloproteinase-2, -7, -9 and -12. This study focuses on investigating the elastinolytic capability of membrane-type 1 matrix metalloproteinase, also known as matrix metalloproteinase-14. We digested recombinant human tropoelastin and human skin elastin with matrix metalloproteinase-14 and analyzed the peptide mixtures using complementary mass spectrometric techniques and bioinformatics tools. The results and additional molecular docking studies show that matrix metalloproteinase-14 cleaves tropoelastin as well as elastin. While tropoelastin was well degraded, fewer cleavages occurred in the highly cross-linked mature elastin. The study also provides insights into the cleavage preferences of the enzyme. Similar to cleavage preferences of matrix metalloproteinases-2, -7, -9 and -12, matrix metalloproteinase-14 prefers small and medium-sized hydrophobic residues including Gly, Ala, Leu and Val at cleavage site P1'. Pro, Gly and Ala were preferably found at P1-P4 and P2'-P4' in both tropoelastin and elastin. Cleavage of mature skin elastin by matrix metalloproteinase-14 released a variety of bioactive elastin peptides, which indicates that the enzyme may play a role in the development and progression of cardiovascular diseases that go along with elastin breakdown.

Elastin is an essential structural protein in the extracellular matrix of vertebrates. It is the core component of elastic fibers, which enable connective tissues such as those of the skin, lungs or blood vessels to stretch and recoil. This function is provided by elastin's exceptional properties, which mainly derive from a unique covalent cross-linking between hydrophilic lysine-rich motifs of units of the monomeric precursor tropoelastin. To date, elastin's cross-linking is poorly investigated. Here, we purified elastin from human tissue and cleaved it into soluble peptides using proteases with different specificities. We then analyzed elastin's molecular structure by identifying unmodified residues, post-translational modifications and cross-linked peptides by high-resolution mass spectrometry and amino acid analysis. The data revealed the presence of multiple isoforms in parallel and a complex and heterogeneous molecular interconnection. We discovered that the same lysine residues in different monomers were simultaneously involved in various cross-link types or remained unmodified. Furthermore, both types of cross-linking domains, Lys-Pro and Lys-Ala domains, participate not only in bifunctional inter- but also in intra-domain cross-links. We elucidated the sequences of several desmosine-containing peptides and the contribution of distinct domains such as 6, 14 and 25. In contrast to earlier assumptions proposing that desmosine cross-links are formed solely between two domains, we elucidated the structure of a peptide that proves a desmosine formation with participation of three Lys-Ala domains. In summary, these results provide new and detailed insights into the cross-linking process, which takes place within and between human tropoelastin units in a stochastic manner. This article is protected by copyright. All rights reserved.

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin's exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and non-cross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure.

Neprilysin is also known as skin fibroblast-derived elastase, and its up-regulation during aging is associated with impairments of the elastic fiber network, loss of skin elasticity and wrinkle formation. However, information on its elastase activity is still limited. The aim of this study was to investigate the degradation of fibrillar skin elastin by neprilysin and the influence of the donor's age on the degradation process using mass spectrometry and bioinformatics approaches. The results showed that cleavage by neprilysin is dependent on previous damage of elastin. While neprilysin does not cleave young and intact skin elastin well, it degrades elastin fibers from older donors, which may further promote aging processes. With regards to the cleavage behavior of neprilysin, a strong preference for Gly at P1 was found, while Gly, Ala and Val were well accepted at P1' upon cleavage of tropoelastin and skin elastin. The results of the study indicate that the progressive release of bioactive elastin peptides by neprilysin upon skin aging may enhance local tissue damage and accelerate extracellular matrix aging processes.

Elastin is a vital protein of the extracellular matrix of jawed vertebrates and provides elasticity to numerous tissues. It is secreted in the form of its soluble precursor tropoelastin, which is subsequently cross-linked in the course of the elastic fiber assembly. The process involves the formation of the two tetrafunctional amino acids desmosine (DES) and isodesmosine (IDES), which are unique to elastin. The resulting high degree of cross-linking confers remarkable properties, including mechanical integrity, insolubility, and long-term stability to the protein. These characteristics hinder the structural elucidation of mature elastin. However, MS(2) data of linear and cross-linked peptides released by proteolysis can provide indirect insights into the structure of elastin. In this study, we performed energy-resolved collision-induced dissociation experiments of DES, IDES, their derivatives, and DES-/IDES-containing peptides to determine characteristic product ions. It was found that all investigated compounds yielded the same product ion clusters at elevated collision energies. Elemental composition determination using the exact masses of these ions revealed molecular formulas of the type CxHyN, suggesting that the pyridinium core of DES/IDES remains intact even at relatively high collision energies. The finding of these specific product ions enabled the development of a similarity-based scoring algorithm that was successfully applied on LC-MS/MS data of bovine elastin digests for the identification of DES-/IDES-cross-linked peptides. This approach facilitates the straightforward investigation of native cross-links in elastin.

This study aimed to characterize the structures of two elastin-like constructs, one composed of a cross-linked elastin-like polypeptide and the other one of cross-linked tropoelastin, and native aortic elastin. The structures of the insoluble materials and human aortic elastin were investigated using scanning electron microscopy. Additionally, all samples were digested with enzymes of different specificities, and the resultant peptide mixtures were characterized by ESI mass spectrometry and MALDI mass spectrometry. The MS(2) data was used to sequence linear peptides, and cross-linked species were analyzed with the recently developed software PolyLinX. This enabled the identification of two intramolecularly cross-linked peptides containing allysine aldols in the two constructs. The presence of the tetrafunctional cross-link desmosine was shown for all analyzed materials and its quantification revealed that the cross-linking degree of the two in vitro cross-linked materials was significantly lower than that of native elastin. Molecular dynamics simulations were performed, based on molecular species identified in the samples, to follow the formation of elastin cross-links. The results provide evidence for the significance of the GVGTP hinge region of domain 23 for the formation of elastin cross-links. Overall, this work provides important insight into structural similarities and differences between elastin-like constructs and native elastin. Furthermore, it represents a step toward the elucidation of the complex cross-linking pattern of mature elastin.