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Cellular senescence leads to the functional decline of regenerative cells such as mesenchymal stromal/stem cells (MSCs), which gives rise to chronic conditions and contributes to poor cell therapy outcomes. Aging tissues are associated with extracellular matrix (ECM) dysregulation, including loss of elastin. However, the role of the ECM in modulating senescence is underexplored. In this work, it is shown that tropoelastin, the soluble elastin precursor, is not only a marker of young MSCs but also actively preserves cell fitness and delays senescence during replicative aging. MSCs briefly exposed to tropoelastin exhibit upregulation of proliferative genes and concurrent downregulation of senescence genes. The seno‐protective benefits of tropoelastin persist during continuous, long‐term MSC culture, and significantly extend the MSC replicative lifespan. Tropoelastin‐expanded MSCs further maintain youth‐associated phenotype and function compared to age‐matched controls, including preserved clonogenic potential, minimal senescence‐associated beta‐galactosidase activity, maintained cell sizes, reduced expression of senescence markers, suppressed secretion of senescence‐associated factors, and increased production of youth‐associated proteins. This work points to the utility of exogenously‐supplemented tropoelastin for manufacturing MSCs that robustly maintain regenerative potential with age. It further reveals the active role of classical structural ECM proteins in driving cellular age‐associated fitness, potentially leading to future interventions for aging‐related pathologies. [ABSTRACT FROM AUTHOR]

Synthetic vascular grafts are commonly used in patients with severe occlusive arterial disease when autologous grafts are not an option. Commercially available synthetic grafts are confronted with challenging outcomes: they have a lower patency rate than autologous grafts and are currently unable to promote arterial regeneration. Polyglycerol sebacate (PGS), a non‐toxic polymer with a tunable degradation profile, has shown promising results as a small‐diameter vascular graft component that can support the formation of neoarteries. In this review, we first present an overview of the synthesis and modification of PGS followed by an examination of its mechanical properties. We then report on the performance, degradation, regeneration, and remodeling of PGS‐based small‐diameter vascular grafts, with a focus on efforts to reduce thrombosis, prevent dilation, and promote cellular residency and extracellular matrix regeneration that resembles the native artery in spatial distribution and organization. We also highlight recent advances in the incorporation of novel in situ cell sources for arterial regeneration and their potential application in PGS‐based vascular grafts. Finally, we compare vascular grafts fabricated using PGS‐based materials with other elastomeric alternatives. [ABSTRACT FROM AUTHOR]

Elastin is an extracellular matrix component with key structural and biological roles in elastic tissues. Interactions between resident cells and tropoelastin, the monomer of elastin, underpin elastin's regulation of cellular processes. However, the nature of tropoelastin–cell interactions and the contributions of individual tropoelastin domains to these interactions are only partly elucidated. In this study, we identified and characterized novel cell‐adhesive sites in the tropoelastin N‐terminal region between domains 12 and 16. We found that this region interacts with αV and α5β1 integrin receptors, which mediate cell attachment and spreading. A peptide sequence from within this region, spanning domains 14 to mid‐domain 16, binds heparan sulfate through electrostatic interactions with peptide lysine residues and induces conformational ordering of the peptide. We propose that domains 14–16 direct initial cell attachment through cell‐surface heparan sulfate glycosaminoglycans, followed by αV and α5β1 integrin‐promoted attachment and spreading on domains 12–16 of tropoelastin. These findings advance our mechanistic understanding of elastin matrix biology, with the potential to enhance tissue regenerative outcomes of elastin‐based materials. [ABSTRACT FROM AUTHOR]

Tropoelastin is the dominant monomer that assembles to form elastin, which confers elasticity to vertebrate elastic tissues including skin, arteries, and lungs. Tropoelastin interacts with cells through cell surface receptors including integrins and glycosaminoglycans (GAGs). As the region 17-18 is recognized as a key region in cell attachment and spreading, we utilized C-terminal truncated tropoelastin constructs containing dissected sections of domain 18. We mapped a cell-interactive sequence of tropoelastin to domain 17 and the first six amino acids (aa) of domain 18. Further delineation identified a 21-residue sequence (Peptide 302-322) which promoted cell attachment and spreading indistinguishable from that to N18, a construct encompassing the full domains 17-18. Alanine substitution of the lysines at positions 11 and 14 in Peptide 302-322 effectively abolished cell binding. This reliance on lysines pointed to a role for GAGs, which was assessed by heparan sulfate inhibition, leading to 85.9 ± 4.2% decreased cell binding, while inhibition of integrins using ethylenediaminetetraacetic acid did not affect attachment. In contrast, selective antibody blocking of the integrin αv family prevented cell spreading by 92.5 ± 8.9%. We propose a two-step mechanism by which cell interactions occur at this central region of tropoelastin: initially, cell adhesion is mediated by GAGs, which contact the lysine residues within the target sequence, and subsequently facilitate cell spreading modulated by integrins, specifically αvβ3 and αvβ5. We conclude that this region comprises a tropoelastin-derived, cell-interactive sequence that independently mediates potent cell binding and spreading via sequential recognition of GAG and integrin cell surface receptors. [ABSTRACT FROM AUTHOR]

Heart failure is a major international health issue. Myocardial mass loss and lack of contractility are precursors to heart failure. Surgical demand for effective myocardial repair is tempered by a paucity of appropriate biological materials. These materials should conveniently replicate natural human tissue components, convey persistent elasticity, promote cell attachment, growth and conformability to direct cell orientation and functional performance. Here, microfabrication techniques are applied to recombinant human tropoelastin, the resilience-imparting protein found in all elastic human tissues, to generate photocrosslinked biological materials containing well-defined micropatterns. These highly elastic substrates are then used to engineer biomimetic cardiac tissue constructs. The micropatterned hydrogels, produced through photocrosslinking of methacrylated tropoelastin (MeTro), promote the attachment, spreading, alignment, function, and intercellular communication of cardiomyocytes by providing an elastic mechanical support that mimics their dynamic mechanical properties in vivo. The fabricated MeTro hydrogels also support the synchronous beating of cardiomyocytes in response to electrical field stimulation. These novel engineered micropatterned elastic gels are designed to be amenable to 3D modular assembly and establish a versatile, adaptable foundation for the modeling and regeneration of functional cardiac tissue with potential for application to other elastic tissues. [ABSTRACT FROM AUTHOR]

Tunable protein composites are important for constructing extracellular matrix mimics of human nerve tissues with control of charge, structural, and mechanical properties. Molecular interaction mechanisms between silk fibroin protein and recombinant human tropoelastin, based on charge, are utilized to generate a new group of multifunctional protein alloys with different net charges. These new biomaterials are then utilized as a biomaterial platform to control neuron cell response. With a +38 net charge in water, tropoelastin molecules provide extraordinary elasticity and selective interactions with cell surface integrins. In contrast, negatively charged silk fibroin protein (net charge −36) provides remarkable toughness and stiffness with morphologic stability in material formats via autoclaving-induced beta-sheet crystal physical crosslinks. The combination of these properties in alloy format extends the versatility of both structural proteins, providing a new biocompatible, biodegradable, and charge-tunable biomaterial platform for neural repair. The data point to these protein alloys as an alternative to commonly used charged synthetic polymers, particularly with regard to the versatility of material formats (e.g., gels, sponges, films, fibers). The results also provide a practical example of physically designed protein materials with control of net charge to direct biological outcomes, in this case for neuronal tissue engineering. [ABSTRACT FROM AUTHOR]

New multifunctional, degradable, polymeric biomaterial systems would provide versatile platforms to address cell and tissue needs in both in vitro and in vivo environments. While protein-based composites or alloys are the building blocks of biological organisms, similar systems have not been largely exploited, to date, to generate ad hoc biomaterials able to control and direct biological functions by recapitulating their inherent structural and mechanical complexities. Therefore, we have recently proposed silktropoelastin material platforms that are able to conjugate a mechanically robust and durable protein, silk, to a highly flexible and biologically active protein, tropoelastin. This review focuses on the elucidation of the interactions between silk and tropoelastin to control the structure of the material its properties, and ultimately functions. In addition, an approach is provided for novel material designs to provide tools to control biological outcomes through surface roughness, elasticity, and net charge for neuronal and mesenchymal stem-cell-based tissue engineering. [ABSTRACT FROM AUTHOR]

Following penetrating injury of the skin, a highly orchestrated and overlapping sequence of events helps to facilitate wound resolution. Inflammation is a hallmark that is initiated early, but the reciprocal relationship between cells and matrix molecules that triggers and maintains inflammation is poorly appreciated. Elastin is enriched in the deep dermis of skin. We propose that deep tissue injury encompasses elastin damage, yielding solubilized elastin that triggers inflammation. As dermal fibroblasts dominate the deep dermis, this means that a direct interaction between elastin sequences and fibroblasts would reveal a proinflammatory signature. Tropoelasfin was used as a surrogate for elasfin sequences. Tropoelastin triggered fibroblast expression of the metalloelastase MMP-12, which is normally expressed by macrophages. MMP-12 expression increased 1056 ± 286-fold by 6 h and persisted for 24 h. Chemokine expression was more transient, as chemokine C-X-C motif ligand 8 (CXCL8), CXCL1, and CXCL5 transcripts increased 11.8 ± 2.6-, 10.2 ± 0.4-, and 8593 ± 996-fold, respectively, by 6-12 h and then decreased. Through the use of specific inhibitors and protein truncation, we found that transduction of the tropoelaslin signal was mediated by the fibroblast elastin binding protein (EBP). In silico modeling using a predictive computational fibroblast model confirmed the up-regulation, and simulations revealed PKA as a key part of the signaling circuit. We tested this prediction with 1 µM PKA inhibitor H-89 and found that 2 h of exposure correspondingly reduced expression of MMP-12 (63.9±12.3%) and all chemokine markers, consistent with the levels seen with EBP inhibition, and validated PKA as a novel node and druggable target to ameliorate the proinflammatory state. A separate trigger that utilized C-terminal RKRK of tropoelasfin reduced marker expression to 65.0-76.5% and suggests the parallel involvement of integrin αvβ3. We propose that the solubilization of elasfin as a result of dermal damage leads to rapid chemokine up-regulation by fibroblasts that is quenched when exposed elasfin is removed by MMP-12. [ABSTRACT FROM AUTHOR]

We generated parallel elastic fibers from synthetic elastin (SE) as a model of the arterial media and assessed the alignment of smooth muscle cells (SMCs). SE utilized crosslinked electrospun human tropoelastin to form aligned fibers that mimicked the topography and elastin-rich content of the medial extracellular matrix. Bundled parallel fibers were anisotropically more elastic than randomly arranged scaffolds (111 ± 25 kPa vs. 265 ± 17 kPa) in the direction of the fibers. Aligned and random fiber scaffolds each supported SMC growth. Following attachment, SMCs proliferated longitudinally on the parallel fibers and expressed native α-smooth muscle actin. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012. [ABSTRACT FROM AUTHOR]

To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site specificities of the enzymes using complementary MS techniques and molecular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty-nine cleavage sites in tropoelastin were identified for MMP-12, whereas MMP-7 and MMP-9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N-terminal and C-terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and/or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P1′. MMP-7 shows a strong preference for Leu at P1′, which is also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12 best tolerates bulky charged and aromatic amino acids at P1′. All three MMPs showed a clear preference for Pro at P3 that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP-12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing. Structured digital abstract • : MMP-7 (uniprotkb: ) cleaves ( ) Tropoelastin (uniprotkb: ) by protease assay ( ) • : MMP-9 (uniprotkb: ) cleaves ( ) Tropoelastin (uniprotkb: ) by protease assay ( ) • : MMP-12 (uniprotkb: ) cleaves ( ) Tropoelastin (uniprotkb: ) by protease assay ( ) [ABSTRACT FROM AUTHOR]

Microfibrils and elastin are major constituents of elastic fibers, the assembly of which is dictated by multimolecular interactions. Microfibril-associated glycoprotein-1 (MAGP-1) is a microfibrillar component that interacts with the soluble elastin precursor, tropoelastin. We describe here the adaptation of a solid-phase binding assay that defines the effect of divalent cations on the interactions between MAGP-1 and tropoelastin. Using this assay, a strong calcium-dependent interaction was demonstrated, with a dissociation constant of 2.8 ± 0.3 n m, which fits a single-site binding model. Manganese and magnesium bestowed a weaker association, and copper did not facilitate the protein interactions. Three constructs spanning tropoelastin were used to quantify their relative contributions to calcium-dependent MAGP-1 binding. Binding to a construct spanning a region from the N-terminus to domain 18 followed a single-site binding model with a dissociation constant of 12.0 ± 2.2 n m, which contrasted with the complex binding behavior observed for fragments spanning domains 17–27 and domain 27 to the C-terminus. To further elucidate binding sites around the kallikrein cleavage site of domains 25/26, MAGP-1 was presented with constructs containing C-terminal deletions within the region. Construct M1659, which spans a region from the N-terminus of tropoelastin to domain 26, inclusive, bound MAGP-1 with a dissociation constant of 9.7 ± 2.0 n m, which decreased to 4.9 ± 1.0 n m following the removal of domain 26 (M155n), thus displaying only half the total capacity to bind MAGP-1. These results demonstrate that MAGP-1 is capable of cumulative binding to distinct regions on tropoelastin, with different apparent dissociation constants and different amounts of bound protein. [ABSTRACT FROM AUTHOR]

Examines the deficient coacervation of two forms of human tropoelastin associated with supravalvular aortic stenosis. Formation of elastic fiber; Provision of elastic properties to artery; Assembly of tropoelastin.

Proton NMR studies have been performed on a 9.8-kDa synthetic fragment comprising the homodimeric leucine zipper domain of the human oncoprotein Jun lo ascertain its conformation in aqueous solution. Analysis of two-dimensional scalar and dipolar-coupling experiments enabled almost all proton resonances to be sequence-specifically assigned and further revealed that the Jun leucine zipper forms a completely symmetric alimet in solution, consistent with the formation of a coiled-coil arrangement of parallel α-helical strands. The rates of exchange of individual amide protons with solvent, as well as hydrogen-bond lengths predicted from amide proton chemical shifts, are shown to correlate with residue position in the coiled-coil. A subset of 209 unambiguous distance constraints was compiled using rules recently formulated for interpreting the NOESY spectra of symmetric coiled-coils, and these were used in combination with experimentally determined hydrogen bond and dihedral angle constraints to compute a solution structure for the Jun leucine zipper domain. [ABSTRACT FROM AUTHOR]

Elastic fibres are an important component of the extracellular matrix and are made of two major components : the more abundant cross-linked elastic protein elastin and the multi-component microfibrils. The biosynthesis of elastic fibres is a complex process involving the interplay of many diverse proteins and genes with elastin as the major component. Tropoelastin is the soluble precursor of elastin and as such it plays a dominant role in elastogenesis. The expression of tropoelastin is under a complex control mechanism, with many isoforms existing. Numerous other components, including the microfibrillar proteins, the elastin-binding protein and lysyl oxidase, the enzyme which initiates elastin cross-linking, are involved in elastogenesis. Tropoelastin undergoes self-association under physiological conditions in a process referred to as coacervation, and this is thought to be a vital process during elastic fibre formation and in providing elasticity. Although various models explaining the elasticity of elastin have been put forward, only the fibrillar model is based on the coacervation ability of tropoelastin. With the molecular cloning of a number of components of the elastic fibre, the availability of these components is increasing and paves the way for in vitro modelling of complex interactions of the elastic fibre. This review emphasises the biochemistry of tropoelastin and its role in elastic fibre structure and assembly. [ABSTRACT FROM AUTHOR]

Coacervation of soluble tropoelastin molecules is characterized by thermodynamically reversible association as temperature is increased under appropriately juxtaposed ionic conditions, protein concentration and pH. Coacervation plays a critical role in the assembly of these elastin precursors in elastic fiber formation. To examine the effect of physiological parameters on the ability of tropoelastin molecules to associate, solutions of recombinant human tropoelastin were monitored spectrophotometrically by light scattering over a broad range of temperatures. Coacervation of recombinant human tropoelastin is strongly influenced by the concentration of protein and NaCl and to a lesser extent on pH. Trends towards maximal association are apparent when each of these parameters is varied. Remarkably, optimal coacervation is found at 37°C, 150 mM NaCl and pH 7–8. Using the data generated by time courses, estimates of thermodynamic parameters were made. These estimates confirm that coacervation is endothermic and is marked by a strong entropic contribution. Circular dichroism of recombinant human tropoelastin revealed that, rather than being random, the structure is compatible with being largely that, of an all-β protein (with secondary structure estimated to be 3% α-helix, 41% β-sheet, 21% β-turn and 33 % other), exhibiting a spectrum as previously seen for tropoelastin populations and soluble elastin from naturally-derived sources. [ABSTRACT FROM AUTHOR]

Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of c-Jun plus a total of five engineered non-native residues at the N- and C-termini. ThejunLZ gene was cloned into the pGEX2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (≃15% total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carboxy-terminal portion of Schistosoma japonicum glutathione Stransferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly [SUP15]N-iabelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel a-helices. We also present [SUP15]N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy. [ABSTRACT FROM AUTHOR]

Tropoelastin is crosslinked with methacrylation and light to make MeTro. MeTro enables encapsulation and surface interactions with cells, as shown by A. Khademhosseini, A. S. Weiss, and co‐workers on page 4950. Patterned MeTro is elastic and supports the organized growth of functionally active, beating cardiomyocytes. Immunostaining shows α‐sarcomeric actinin (green), connexin‐43 (red), and nuclei (blue). [ABSTRACT FROM AUTHOR]