Human Tropoelastin Research Articles

Sequence and domain arrangements influence mechanical properties of elastin‐like polymeric elastomers

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self-assembly that aligns lysine residues for crosslinking. As a result, both the full-length monomer as well as elastin-like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full-length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self-assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full-length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties.

Charge-Tunable Silk-Tropoelastin Protein Alloys That Control Neuron Cell Responses.

Tunable protein composites are important for constructing extracellular matrix mimics of human tissues with control of biochemical, 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 (mixtures of silk and tropoelastin) 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 biomaterial platform. The alloys with weak positive charges (silk/tropoelastin mass ratio 75/25, net charge around +16) significantly improved the formation of neuronal networks and maintained cell viability of rat cortical neurons after 10 days in vitro. The data point to these protein alloys as an alternative to commonly used poly-L-lysine (PLL) coatings or other 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.

Elastin, a Novel Extracellular Matrix Protein Adhering to Mycobacterial Antigen 85 Complex

The antigen 85 complex (Ag85) consists of three predominantly secreted proteins (Ag85A, Ag85B, and Ag85C), which play a key role in the mycobacterial pathogenesis and also possess enzymatic mycolyltransferase activity involved in cell wall synthesis. Ag85 is not only considered to be a virulence factor because its expression is essential for intracellular survival within macrophages, but also because it contributes to adherence, invasion, and dissemination of mycobacteria in host cells. In this study, we report that the extracellular matrix components, elastin and its precursor (tropoelastin) derived from human aorta, lung, and skin, serve as binding partners of Ag85 from Mycobacterium tuberculosis. The binding affinity of M. tuberculosis Ag85 to human tropoelastin was characterized (K(D) = 0.13 ± 0.006 μm), and a novel Ag85-binding motif, AAAKAA(K/Q)(Y/F), on multiple tropoelastin modules was identified. In addition, the negatively charged Glu-258 of Ag85 was demonstrated to participate in an electrostatic interaction with human tropoelastin. Moreover, binding of Ag85 on elastin siRNA-transfected Caco-2 cells was significantly reduced (34.3%), implying that elastin acts as an important ligand contributing to mycobacterial invasion.

Characterizing the mechanical properties of tropoelastin protein scaffolds

ABSTRACTThis paper reports on mechanical characterization of electrospun tissue scaffolds formed from varying blends of collagen and human tropoelastin. The electrospun tropoelastin-based scaffolds have an open, porous structure conducive to cell attachment and have been shown to exhibit strong biocompatibility, but the mechanical character is not well known. Mechanical properties were tested for scaffolds consisting of 100% tropoelastin and 1:1 tropoelastin-collagen blends. The results showed that the materials exhibited a three order of magnitude change in the initial elastic modulus when tested dry vs. hydrated, with moduli of 21 MPa and 0.011 MPa respectively. Noncrosslinked and crosslinked tropoelastin scaffolds exhibited the same initial stiffness from 0 to 50% strain, and the noncrosslinked scaffolds exhibited no stiffness at strains >∼50%. The elastic modulus of a 1:1 tropoelastin-collagen blend was 50% higher than that of a pure tropoelastin scaffold. Finally, the 1:1 tropoelastin-collagen blend was five times stiffer from 0 to 50% strain when strained at five times the ASTM standard rate. By systematically varying protein composition and crosslinking, the results demonstrate how protein scaffolds might be manipulated as customized biomaterials, ensuring mechanical robustness and potentially improving biocompatibility through minimization of compliance mismatch with the surrounding tissue environment. Moreover, the demonstration of strain-rate dependent mechanical behavior has implications for mechanical design of tropoelastin-based tissue scaffolds.

Amyloidogenesis of proteolytic fragments of human elastin

Some polypeptides encoded by the C-terminal region of human tropoelastin gene have been demonstrated to be amyloidogenic in vitro. The biological relevance of this finding is still under investigation given that only limited evidence concerning the involvement of elastin in amyloidosis exists. Recent studies identified, by mass spectrometry, several elastin fragments produced from the cleavages made by some elastases in human elastin substrate. Some of these fragments are contained into the same polypeptide sequences previously demonstrated to be amyloidogenic. Our hypothesis is that the up-regulation of elastases in inflammatory processes triggered, for example, by aging induces the release of elastin fragments potentially amyloidogenic. Therefore our aim in this study is to demonstrate if any of these fragments is amyloidogenic in vitro. At molecular level, CD, NMR, FTIR spectroscopies and MD simulations were used, while, at supramolecular level, Congo red binding assay and ThT fluorescence spectroscopy complemented with AFM microscopy were carried out. Our results show that the longest peptide, among those synthesized and mimicking the elastin fragments produced by elastases on human elastin, constituted of 22 residues, is able to aggregate into amyloid-like fibres. These findings support the hypothesis of amyloidogenesis of proteolytic fragments of elastin.

A Tunable and Reversible Platform for the Intracellular Formation of Genetically Engineered Protein Microdomains

From mitochondria to the nuclear envelope, the controlled assembly of micro- and nanostructures is essential for life; however, the level at which we can deliberately engineer the assembly of microstructures within intracellular environments remains primitive. To overcome this obstacle, we present a platform to reversibly assemble genetically engineered protein microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired from the human protein tropoelastin, these protein polymers form a secondary aqueous phase above a tunable transition temperature. This assembly process is easily manipulated to occur at or near physiological temperature by adjusting molecular weight and hydrophobicity. We fused protein polymers to green fluorescent protein (GFP) to visualize their behavior within the cytoplasm. While soluble, these polymers have a similar intracellular diffusion constant as cytosolic proteins at 7.4 μm(2)/s; however, above their phase transition temperature, the proteins form distinct microdomains (0.1-2 μm) with a reduced diffusion coefficient of 1.1 μm(2)/s. Microdomain assembly and disassembly are both rapid processes with half-lives of 3.8 and 1.0 min, respectively. Via selection of the protein polymer, the assembly temperature is tunable between 20 and 40 °C. This approach may be useful to control intracellular formation of genetically engineered proteins and protein complexes into concentrated microdomains.

Polymorphisms in the Human Tropoelastin Gene Modify In Vitro Self-Assembly and Mechanical Properties of Elastin-Like Polypeptides

Elastin is a major structural component of elastic fibres that provide properties of stretch and recoil to tissues such as arteries, lung and skin. Remarkably, after initial deposition of elastin there is normally no subsequent turnover of this protein over the course of a lifetime. Consequently, elastic fibres must be extremely durable, able to withstand, for example in the human thoracic aorta, billions of cycles of stretch and recoil without mechanical failure. Major defects in the elastin gene (ELN) are associated with a number of disorders including Supravalvular aortic stenosis (SVAS), Williams-Beuren syndrome (WBS) and autosomal dominant cutis laxa (ADCL). Given the low turnover of elastin and the requirement for the long term durability of elastic fibres, we examined the possibility for more subtle polymorphisms in the human elastin gene to impact the assembly and long-term durability of the elastic matrix. Surveys of genetic variation resources identified 118 mutations in human ELN, 17 being non-synonymous. Introduction of two of these variants, G422S and K463R, in elastin-like polypeptides as well as full-length tropoelastin, resulted in changes in both their assembly and mechanical properties. Most notably G422S, which occurs in up to 40% of European populations, was found to enhance some elastomeric properties. These studies reveal that even apparently minor polymorphisms in human ELN can impact the assembly and mechanical properties of the elastic matrix, effects that over the course of a lifetime could result in altered susceptibility to cardiovascular disease.

Elastin-Like Peptide Amphiphiles Form Nanofibers with Tunable Length

Peptide amphiphiles (PAs) self-assemble nanostructures with potential applications in drug delivery and tissue engineering. Some PAs share environmentally responsive behavior with their peptide components. Here we report a new type of PAs biologically inspired from human tropoelastin. Above a lower critical solution temperature (LCST), elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition. Similar to other PAs, elastin-like PAs (ELPAs) assemble micelles with fiber-like nanostructures. Similar to ELPs, ELPAs have inverse phase transition behavior. Here we demonstrate control over the ELPAs fiber length and cellular uptake. In addition, we observed that both peptide assembly and nanofiber phase separation are accompanied by a distinctive secondary structure attributed primarily to a type-1 β turn. We also demonstrate increased solubility of hydrophobic paclitaxel (PAX) in the presence of ELPAs. Due to their biodegradability, biocompatibility, and environmental responsiveness, elastin-inspired biopolymers are an emerging platform for drug and cell delivery; furthermore, the discovery of ELPAs may provide a new and useful approach to engineer these materials into stimuli-responsive gels and drug carriers.

Electrospun synthetic human elastin:collagen composite scaffolds for dermal tissue engineering

We present an electrospun synthetic human elastin:collagen composite scaffold aimed at dermal tissue engineering. The panel of electrospun human tropoelastin and ovine type I collagen blends comprised 80% tropoelastin+20% collagen, 60% tropoelastin+40% collagen and 50% tropoelastin+50% collagen. Electrospinning efficiency decreased with increasing collagen content under the conditions used. Physical and mechanical characterization encompassed fiber morphology, porosity, pore size and modulus, which were prioritized to identify the optimal candidate for dermal tissue regeneration. Scaffolds containing 80% tropoelastin and 20% collagen (80T20C) were selected on this basis for further cell interaction and animal implantation studies. 80T20C enhanced proliferation and migration rates of dermal fibroblasts in vitro and were well tolerated in a mouse subcutaneous implantation study where they persisted over 6weeks. The 80T20C scaffolds supported fibroblast infiltration, de novo collagen deposition and new capillary formation.

Biodegradation of elastin‐like polypeptide nanoparticles

Protein polymers are repetitive polypeptides produced by ribosomal biosynthetic pathways; furthermore, they offer emerging opportunities in drug and biopharmaceutical delivery. As for any polymer, biodegradation is one of the most important determinants affecting how a protein polymer can be utilized in the body. This study was designed to characterize the proteolytic biodegradation for a library of protein polymers derived from the human tropoelastin, the Elastin-like polypeptides (ELPs). ELPs are of particular interest for controlled drug delivery because they reversibly transition from soluble to insoluble above an inverse phase transition temperature (T(t)). More recently, ELP block copolymers have been developed that can assemble into micelles; however, it remains unclear if proteases can act on these ELP nanoparticles. For the first time, we demonstrate that ELP nanoparticles can be degraded by two model proteases and that comparable proteolysis occurs after cell uptake into a transformed culture of murine hepatocytes. Both elastase and collagenase endopeptidases can proteolytically degrade soluble ELPs. To our surprise, the ELP phase transition was protective against collagenase but not to elastase activity. These findings enhance our ability to predict how ELPs will biodegrade in different physiological microenvironments and are essential to develop protein polymers into biopharmaceuticals.

Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy

In this study, one- and two-dimensional NMR experiments are applied to uniformly (15)N-enriched synthetic elastin, a recombinant human tropoelastin that has been cross-linked to form an elastic hydrogel. Hydrated elastin is characterized by large segments that undergo "liquid-like" motions that limit the efficiency of cross-polarization. The refocused insensitive nuclei enhanced by polarization transfer experiment is used to target these extensive, mobile regions of this protein. Numerous peaks are detected in the backbone amide region of the protein, and their chemical shifts indicate the completely unstructured, "random coil" model for elastin is unlikely. Instead, more evidence is gathered that supports a characteristic ensemble of conformations in this rubber-like protein.

Molecular Orientation of Tropoelastin is Determined by Surface Hydrophobicity

Tropoelastin is the precursor of the extracellular protein elastin and is utilized in tissue engineering and implant technology by adapting the interface presented by surface-bound tropoelastin. The preferred orientation of the surface bound protein is relevant to biointerface interactions, as the C-terminus of tropoelastin is known to be a binding target for cells. Using recombinant human tropoelastin we monitored the binding of tropoelastin on hydrophilic silica and on silica made hydrophobic by depositing a self-assembled monolayer of octadecyl trichlorosilane. The layered organization of deposited tropoelastin was probed using neutron and X-ray reflectometry under aqueous and dried conditions. In a wet environment, tropoelastin retained a solution-like structure when adsorbed on silica but adopted a brush-like structure when on hydrophobized silica. The orientation of the surface-bound tropoelastin was investigated using cell binding assays and it was found that the C-terminus of tropoelastin faced the bulk solvent when bound to the hydrophobic surface, but a mixture of orientations was adopted when tropoelastin was bound to the hydrophilic surface. Drying the tropoelastin-coated surfaces irreversibly altered these protein structures for both hydrophilic and hydrophobic surfaces.

Enhancement of Biocompatibility of Synthetic Vascular Grafts by Covalent Immobilisation of Recombinant Human Tropoelastin

Enhancement of Biocompatibility of Synthetic Vascular Grafts by Covalent Immobilisation of Recombinant Human Tropoelastin

TCT-184: Immobilized recombinant human tropoelastin on a plasma-activated coating dramatically enhances biocompatibility of metal alloys: implications for coronary stents

TCT-184: Immobilized recombinant human tropoelastin on a plasma-activated coating dramatically enhances biocompatibility of metal alloys: implications for coronary stents

Alignment of human vascular smooth muscle cells on parallel electrospun synthetic elastin fibers

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.

The influence of elasticity and surface roughness on myogenic and osteogenic-differentiation of cells on silk-elastin biomaterials

The interactions of C2C12 myoblasts and human bone marrow stem cells (hMSCs) with silk-tropoelastin biomaterials, and the capacity of each to promote attachment, proliferation, and either myogenic- or osteogenic-differentiation were investigated. Temperature-controlled water vapor annealing was used to control beta-sheet crystal formation to generate insoluble silk-tropoelastin biomaterial matrices at defined ratios of the two proteins. These ratios controlled surface roughness and micro/nano-scale topological patterns, and elastic modulus, stiffness, yield stress, and tensile strength. A combination of low surface roughness and high stiffness in the silk-tropoelastin materials promoted proliferation and myogenic-differentiation of C2C12 cells. In contrast, high surface roughness with micro/nano-scale surface patterns was favored by hMSCs. Increasing the content of human tropoelastin in the silk-tropoelastin materials enhanced the proliferation and osteogenic-differentiation of hMSCs. We conclude that the silk-tropoelastin composition facilitates fine tuning of the growth and differentiation of these cells.

Role of polyproline II conformation in human tropoelastin structure

In this review, we present a comprehensive overview of the molecular studies on human tropoelastin domains accomplished by Tamburro and co-workers in the last decade. The used approach is the reductionist approach applied to human tropoelastin and is based on the observation that the tropoelastin gene exhibits a cassette-like organization, with a regular alternation of cross-linking and hydrophobic domains putatively responsible for the elasticity of the protein. The peculiar structure of human tropoelastin gene prompted us to study the isolated domains encoded by the exons of tropoelastin, with the perspective to get deep insights into the structural properties of the whole protein. At the molecular level, the results clearly evidence large flexibility of the polypeptide chains in the hydrophobic domains, which oscillate between rather extended and folded conformations. An important role was assigned to poly-proline II conformation considered as the hinge structure in the dynamic conformational equilibrium suggested for the hydrophobic domains. For the lysine-rich cross-linking domains, the structural studies exactly localized α-helix along the polypeptide sequence. Furthermore, at supramolecular level, these studies showed that several domains are able to self-assemble in two different aggregation patterns, the fibrous elastin-like structure for some proline-rich hydrophobic domains and the amyloid-like for some glycine-rich hydrophobic domains. Accordingly, the studies suggest that the reductionist approach was a valid tool for studying a complex protein, such as elastin, elucidating not only the structure but also the specific role played by its constituent domains.

Mechanical property characterization of electrospun recombinant human tropoelastin for vascular graft biomaterials

The development of vascular grafts has focused on finding a biomaterial that is non-thrombogenic, minimizes intimal hyperplasia, matches the mechanical properties of native vessels and allows for regeneration of arterial tissue. In this study, the structural and mechanical properties and the vascular cell compatibility of electrospun recombinant human tropoelastin (rTE) were evaluated as a potential vascular graft support matrix. Disuccinimidyl suberate (DSS) was used to cross-link electrospun rTE fibers to produce a polymeric recombinant tropoelastin (prTE) matrix that is stable in aqueous environments. Tubular 1cm diameter prTE samples were constructed for uniaxial tensile testing and 4mm small-diameter prTE tubular scaffolds were produced for burst pressure and cell compatibility evaluations from 15wt.% rTE solutions. Uniaxial tensile tests demonstrated an average ultimate tensile strength (UTS) of 0.36±0.05MPa and elastic moduli of 0.15±0.04 and 0.91±0.16MPa, which were comparable to extracted native elastin. Burst pressures of 485±25mm Hg were obtained from 4mm internal diameter scaffolds with 453±74μm average wall thickness. prTE supported endothelial cell growth with typical endothelial cell cobblestone morphology after 48h in culture. Cross-linked electrospun rTE has promising properties for utilization as a vascular graft biomaterial with customizable dimensions, a compliant matrix and vascular cell compatibility.

Structural and cellular characterization of electrospun recombinant human tropoelastin biomaterials

An off-the-shelf vascular graft biomaterial for vascular bypass surgeries is an unmet clinical need. The vascular biomaterial must support cell growth, be non-thrombogenic, minimize intimal hyperplasia, match the structural properties of native vessels, and allow for regeneration of arterial tissue. Electrospun recombinant human tropoelastin (rTE) as a medial component of a vascular graft scaffold was investigated in this study by evaluating its structural properties, as well as its ability to support primary smooth muscle cell adhesion and growth. rTE solutions of 9, 15, and 20 wt% were electrospun into sheets with average fiber diameters of 167 ± 32, 522 ± 67, and 735 ± 270 nm, and average pore sizes of 0.4 ± 0.1, 5.8 ± 4.3, and 4.9 ± 2.4 µm, respectively. Electrospun rTE fibers were cross-linked with disuccinimidyl suberate to produce an insoluble fibrous polymeric recombinant tropoelastin (prTE) biomaterial. Smooth muscle cells attached via integrin binding to the rTE coatings and proliferated on prTE biomaterials at a comparable rate to growth on prTE coated glass, glass alone, and tissue culture plastic. Electrospun tropoelastin demonstrated the cell compatibility and design flexibility required of a graft biomaterial for vascular applications.

Stability of a Therapeutic Layer of Immobilized Recombinant Human Tropoelastin on a Plasma-Activated Coated Surface

To modify blood-contacting stainless surfaces by covalently coating them with a serum-protease resistant form of tropoelastin (TE). To demonstrate that the modified TE retains an exposed, cell-adhesive C-terminus that persists in the presence of blood plasma proteases. Recombinant human TE and a point mutant variant (R515A) of TE were labeled with (125)Iodine and immobilized on plasma-activated stainless steel (PAC) surfaces. Covalent attachment was confirmed using rigorous detergent washing. As kallikrein and thrombin dominate the serum degradation of tropoelastin, supraphysiological levels of these proteases were incubated with covalently bound TE and R515A, then assayed for protein levels by radioactivity detection. Persistence of the C-terminus was assessed by ELISA. TE was significantly retained covalently on PAC surfaces at 88 ± 5% and 71 ± 5% after treatment with kallikrein and thrombin, respectively. Retention of R515A was 100 ± 1.3% and 87 ± 2.3% after treatment with kallikrein and thrombin, respectively, representing significant improvements over TE. The functionally important C-terminus was cleaved in wild-type TE but retained by R515A. Protein persists in the presence of human kallikrein and thrombin when covalently immobilized on metal substrata. R515A displays enhanced protease resistance and retains the C-terminus presenting a protein interface that is viable for blood-contacting applications.