The protein elastin imparts extensibility, elastic recoil, and resilience to tissues including arterial walls, skin, lung alveoli, and the uterus. Elastin and elastin-like peptides are hydrophobic, disordered, and undergo liquid-liquid phase separation upon self-assembly. Despite extensive study, the structure of elastin remains controversial. We use molecular dynamics simulations on a massive scale to elucidate the structural ensemble of aggregated elastin-like peptides. Consistent with the entropic nature of elastic recoil, the aggregated state is stabilized by the hydrophobic effect. However, self-assembly does not entail formation of a hydrophobic core. The polypeptide backbone forms transient, sparse hydrogen-bonded turns and remains significantly hydrated even as self-assembly triples the extent of non-polar side chain contacts. Individual chains in the assembly approach a maximally-disordered, melt-like state which may be called the liquid state of proteins. These findings resolve long-standing controversies regarding elastin structure and function and afford insight into the phase separation of disordered proteins.

Elastin is one of the most important and abundant extracellular matrix (ECM) proteins that provide elasticity and resilience to tissues and organs, including vascular walls, ligaments, skin, and lung. Besides hereditary diseases, such as Williams-Beuren syndrome (WBS), which results in reduced elastin synthesis, injuries, aging, or acquired diseases can lead to the degradation of existing elastin fibers. Thus, the de novo synthesis of elastin is required in several medical conditions to restore the elasticity of affected tissues. Here, we applied synthetic modified mRNA encoding tropoelastin (TE) for the de novo synthesis of elastin and determined the mRNA-mediated elastin synthesis in cells, as well as ex vivo in porcine skin. EA.hy926 cells, human fibroblasts, and mesenchymal stem cells (MSCs) isolated from a patient with WBS were transfected with 2.5 μg TE mRNA. After 24 hr, the production of elastin was analyzed by Fastin assay and dot blot analyses. Compared with untreated cells, significantly enhanced elastin amounts were detected in TE mRNA transfected cells. The delivered synthetic TE mRNA was even able to significantly increase the elastin production in elastin-deficient MSCs. In porcine skin, approximately 20% higher elastin amount was detected after the intradermal delivery of synthetic mRNA by microinjection. In this study, we demonstrated the successful applicability of synthetic TE encoding mRNA to produce elastin in elastin-deficient cells as well as in skin. Thus, this auspicious mRNA-based integration-free method has a huge potential in the field of regenerative medicine to induce de novo elastin synthesis, e.g., in skin, blood vessels, or alveoli.

This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues.

Biomolecular condensates are macromolecular complexes formed by liquid-liquid phase separation. They regulate key biological functions by reversibly compartmentalizing molecules in cells, in a stimulus-dependent manner. Designing stimuli-responsive synthetic condensates is crucial for engineering compartmentalized synthetic cells that are able to mimic spatiotemporal control over the biochemical reactions. Here, we design and test a family of condensate-forming, pH-responsive elastin-like polypeptides (ELPs) that form condensates above critical pH values ranging between 4 and 7, for temperatures between 20 and at 37 °C. We show that the condensation occurs rapidly, in sharp pH intervals (ΔpH < 0.3). For eventual applications in engineering synthetic cell compartments, we demonstrate that multiple types of pH-responsive ELPs can form mixed condensates inside micron-sized vesicles. When genetically fused with enzymes, receptors, and signaling molecules, these pH-responsive ELPs could be potentially used as pH-switchable functional condensates for spatially controlling biochemistry in engineered synthetic cells.

Liquid-liquid phase separation of tropoelastin has long been considered to be an important early step in the complex process of elastin fiber assembly in the body and has inspired the development of elastin-like peptides with a wide range of industrial and biomedical applications. Despite decades of study, the material state of the condensed liquid phase of elastin and its subsequent maturation remain poorly understood. Here, using a model minielastin that mimics the alternating domain structure of full-length tropoelastin, we examine the elastin liquid phase. We combine differential interference contrast (DIC), fluorescence, and scanning electron microscopy with particle-tracking microrheology to resolve the material transition occurring within elastin liquids over time in the absence of exogenous cross-linking. We find that this transition is accompanied by an intermediate stage marked by the coexistence of insoluble solid and dynamic liquid phases giving rise to significant spatial heterogeneities in material properties. We further demonstrate that varying the length of the terminal hydrophobic domains of minielastins can tune the maturation process. This work not only resolves an important step in the hierarchical assembly process of elastogenesis but further contributes mechanistic insight into the diverse repertoire of protein condensate maturation pathways with emerging importance across biology.

Elastin-derived peptides are released from the extracellular matrix remodeling by numerous proteases and seem to regulate many biological processes, notably cancer progression. The canonical elastin peptide is VGVAPG, which harbors the XGXXPG consensus pattern, allowing interaction with the elastin receptor complex located at the surface of cells. Besides these elastokines, another class of peptides has been identified. This group of bioactive elastin peptides presents the XGXPGXGXG consensus sequence, but the reason for their bioactivity remains unexplained. To better understand their nature and structure-function relationships, herein we searched the current databases for this nonapeptide motif and observed that the XGXPGXGXG elastin peptides define a specific group of tandemly repeated patterns. Further, we focused on four tandemly repeated human elastin nonapeptides, i.e., AGIPGLGVG, VGVPGLGVG, AGVPGLGVG, and AGVPGFGAG. These peptides were analyzed by means of optical spectroscopies and molecular dynamics. Ultraviolet-circular dichroism and Raman spectra are consistent with a mixture of β-turn, β-strand, and random-chain secondary elements in aqueous media. Quantitative analysis of their conformations suggested that turns corresponded to half of the total population of structural elements, whereas the remaining half were equally distributed between β-strand and unordered chains. These distributions were confirmed by molecular dynamics simulations. Altogether, our data suggest that these highly dynamic peptides harbor a type II β-turn located in their central part. We hypothesize that this structural element could explain their specific bioactivity.

Silk-elastin block copolymers have such physical and biological properties that make them attractive biomaterials for applications ranging from tissue regeneration to drug delivery. Silk-elastin block copolymers that only assemble into fibrils at high concentrations can be used for a template-induced fibril assembly. This can be achieved by additionally including template-binding blocks that promote high local concentrations of polymers on the template, leading to a template-induced fibril assembly. We hypothesize that template-inducible silk-fibril formation, and hence high critical concentrations for fibril formation, requires careful tuning of the block lengths, to be close to a critical set of block lengths that separates fibril forming from nonfibril forming polymer architectures. Therefore, we explore herein the impact of tuning block lengths for silk-elastin diblock polypeptides on fibril formation. For silk-elastin diblocks ES m -SQ n , in which the elastin pentamer repeat is ES = GSGVP and the crystallizable silk octamer repeat is SQ = GAGAGAGQ, we find that no fibril formation occurs for n = 6 but that the n = 10 and 14 diblocks do show concentration-dependent fibril formation. For n = 14 diblocks, no effect is observed of the length m (with m = 40, 60, 80) of the amorphous block on the lengths of the fibrils. In contrast, for the n = 10 diblocks that are closest to the critical boundary for fibril formation, we find that long amorphous blocks (m = 80) oppose the growth of fibrils at low concentrations, making them suitable for engineering template-inducible fibril formation.

Desmosine crosslinks are responsible for the elastic properties of connective tissues in lungs and cardiovascular system and are often compromised in disease states. We developed a new, fast, and simple cation exchange HPLC assay for the analysis of desmosine and isodesmosine in animal elastin. The method was validated by determining linearity, accuracy, precision, and desmosines stability and was applied to measure levels of desmosines in porcine and murine organs. The detection and quantification limits were 2 and 4 pmol, respectively. The run-time was 8 min. Our cation exchange column does not separate desmosine and isodesmosine, but their level can be quantified from absorbance at different wavelengths. Using this assay, we found that desmosines levels were significantly lower in elastin isolated from various organs of immunodeficient severe combined immunodeficiency mice compared with wild-type animals. We also found that desmosines levels were lower in lung elastin isolated from hyperhomocysteinemic Pcft(-/-) mice deficient in intestinal folate transport compared with wild-type Pcft(+/+) animals.

Globally, increasing mortality from cardiovascular disease has become a problem in recent years. Vascular replacement has been used as a treatment for these diseases, but with blood vessels <6 mm in diameter, existing vascular grafts made of synthetic polymers can be occluded by thrombus formation or intimal hyperplasia. Therefore, the development of new artificial vascular grafts is desirable. In this study, we developed an elastin (EL)-silk fibroin (SF) double-raschel knitted vascular graft 1.5 mm in diameter. Water-soluble EL was prepared from insoluble EL by hydrolysis with oxalic acid. Compared to SF, EL was less likely to adhere to platelets, while vascular endothelial cells were three times more likely to adhere. SF artificial blood vessels densely packed with porous EL were fabricated, and these prevented the leakage of blood from the graft during implantation, while the migration of cells after implantation was promoted. Several kinds of 13C solid-state NMR spectra were observed with the EL-SF grafts in dry and hydrated states. It was noted that the EL molecules in the graft had very high mobility in the hydrated state. The EL-SF grafts were implanted into the abdominal aorta of rats to evaluate their patency and remodeling ability. No adverse reactions, such as bleeding at the time of implantation or disconnection of the sutured ends, were observed in the implanted grafts, and all were patent at the time of extraction. In addition, vascular endothelial cells were present on the graft's luminal surface 2 weeks after implantation. Therefore, we conclude that EL-SF artificial vascular grafts may be useful where small-diameter grafts are required.

Introduction: Vascular grafts significantly contribute to advances in vascular surgery, but none of the currently available prosthetic grafts have elastin fibers similar to native arteries. We hypothesized that a novel elastin patch could be produced after a rat decellularized thoracic aorta elastin fiber scaffold is implanted subcutaneously in rats; we tested this novel elastin patch in a rat aortic arterioplasty model. Methods: Sprague-Dawley rats (200 g) were used. Rat thoracic aortae were decellularized and sectioned at a thickness of 30 μm. A single elastin fiber scaffold was fabricated as a net (5 × 5 mm2), and then a three-layer scaffold was constructed to make a new patch. The hyaluronic acid-sodium alginate (HA/SA) hydrogel was fabricated by reacting sodium SA, HA, and CaCO3, and then the hydrogel was added to the patch to secure the elastin fibers. The patches were implanted subcutaneously in rats and harvested at day 14. The elastin patches were then implanted into the same rat's aorta and harvested at day 14; a decellularized rat thoracic aorta (TA) patch was used as a control. Sections of the retrieved patches were stained by immunohistochemistry and immunofluorescence. Results: The elastin fibers could be secured by the hydrogel. After 14 days, the subcutaneously implanted elastin patch was incorporated into the rat tissue, and H&E staining showed that new tissue had formed around the elastin patch with almost no hydrogel left. After implantation into the rat aorta and then retrieval on day 14, H&E staining showed that there was neointima and adventitia formation in both the TA and elastin patch groups. Both patches showed a similar histological structure after implantation, and immunofluorescence showed that there were CD34- and nestin-positive cells in the neointima. In both groups, the endothelial cells expressed the arterial identity markers Ephrin-B2 and dll-4; almost one-third of the cells in the neointima were PCNA-positive with rare cleaved caspase-3-positive cells. Conclusion: We demonstrated a novel approach to making elastin fiber scaffold hydrogel patches (elastin patches) and tested them in a rat aorta arterioplasty model. This patch showed a similar healing process as the decellularized TA patch; it also showed potential applications in large animals and may be a substitute for prosthetic grafts in vascular surgery.

Soluble elastin-like peptides (ELPs) can be engineered into a range of physical forms, from hydrogels and scaffolds to fibers and artificial tissues, finding numerous applications in medicine and engineering as "smart polymers". Elastin-like peptides are attractive candidates as a platform for novel biomaterial design because they exhibit a highly tunable response spectrum, with reversible phase transition capabilities. Here, we report the design of the first virtual library of elastin-like protein models using methods for enhanced sampling to study the effect of peptide chemistry, chain length, and salt concentration on the structural transitions of ELPs, exposing associated molecular mechanisms. We describe the behavior of the local molecular structure under increasing temperatures and the effect of peptide interactions with nearest hydration shell water molecules on peptide mobility and propensity to exhibit structural transitions. Shifts in the magnitude of structural transitions at the single-molecule scale are explained from the perspective of peptide-ion-water interactions in a library of four unique elastin-like peptide systems. Predictions of structural transitions are subsequently validated in experiment. This library is a valuable resource for recombinant protein design and synthesis as it elucidates mechanisms at the single-molecule level, paving a feedback path between simulation and experiment for smart material designs, with applications in biomedicine and diagnostic devices.

Williams-Beuren syndrome is the consequence of a large contiguous-gene deletion on the seventh human chromosome that includes the elastin gene. Elastin is an extracellular matrix protein responsible for the cardiovascular abnormalities associated with Williams's syndrome, including hypertension and aortic stenosis. A high percentage of individuals with Williams's syndrome also have impaired glucose tolerance, independent of traditional risk factors for diabetes. Here, we show that murine adipose tissue does assemble elastic fibers; however, isolated elastin insufficiency (Eln+/-) in mice does not independently influence glucose metabolism or tissue lipid accumulation. Similarly, isolated ApoE deficiency (ApoE-/-), a model of hyperlipidemia and atherosclerosis, does not impair insulin sensitivity. However, Eln+/-; ApoE-/- double mutant mice exhibit notable hyperglycemia, adipocyte hypertrophy, inflammation of adipose tissue, and ectopic lipid accumulation in liver tissue. Further, Eln+/-; ApoE-/- mutants have significant impairment of insulin sensitivity by insulin tolerance testing, independent of body weight or diet, suggesting that elastin insufficiency predisposes to metabolic disease in susceptible individuals.

We demonstrate a novel approach for the production of tunable quantities of elastic fibers. We also show that exogenous tropoelastin is rate-limiting for elastin synthesis regardless of the age of the dermal fibroblast donor. Additionally, we provide a strategy to further enhance synthesis by older cells through the application of conditioned media. We show that this approach delivers an elastin layer on one side of the leading dermal repair template for contact with the deep dermis in order to deliver prefabricated elastic fibers to a physiologically appropriate site during subsequent surgery. This system is attractive because it provides for the first time a viable path for sufficient, histologically detectable levels of patient elastin into full-thickness wound sites that have until now lacked this elastic underlayer.

Dermo-epidermal equivalents based on plasma-derived fibrin hydrogels have been extensively studied for skin engineering. However, they showed rapid degradation and contraction over time and low mechanical properties which limit their reproducibility and lifespan. In order to achieve better mechanical properties, elasticity and biological properties, we incorporated a elastin-like recombinamer (ELR) network, based on two types of ELR, one modified with azide (SKS-N3) and other with cyclooctyne (SKS-Cyclo) chemical groups at molar ratio 1:1 at three different SKS (serine-lysine-serine sequence) concentrations (1, 3, and 5 wt.%), into plasma-derived fibrin hydrogels. Our results showed a decrease in gelation time and contraction, both in the absence and presence of the encapsulated human primary fibroblasts (hFBs), higher mechanical properties and increase in elasticity when SKSs content is equal or higher than 3%. However, hFBs proliferation showed an improvement when the lowest SKS content (1 wt.%) was used but started decreasing when increasing SKS concentration at day 14 with respect to the plasma control. Proliferation of human primary keratinocytes (hKCs) seeded on top of the hybrid-plasma hydrogels containing 1 and 3% of SKS showed no differences to plasma control and an increase in hKCs proliferation was observed for hybrid-plasma hydrogels containing 5 wt.% of SKS. These promising results showed the need to achieve a balance between the reduced contraction, the better mechanical properties and biological properties and indicate the potential of using this type of hydrogel as a testing platform for pharmaceutical products and cosmetics, and future work will elucidate their potential.

Among viruses, lentiviral vectors have been popular vectors for gene delivery due to their efficient mode of gene delivery. However, the nonspecific delivery of genes associated with lentiviral vectors may result in undesirable side effects. Here we propose a heterogeneous nanoparticle (NP) delivery system for targeted delivery of lentiviral particles containing a therapeutic gene. The heterogeneous NPs consist of the low-density lipoprotein receptor repeat 3 (LDLR3) and the keratinocyte growth factor (KGF), each fused to elastin-like polypeptides (ELPs), LDLR3-ELP and KGF-ELP, respectively. Our results show that although homogeneous NPs comprising of LDLR3-ELP alone blocked viral transduction, heterogeneous NPs comprising of KGF-ELP and LDLR3-ELP enhanced viral transduction in cells expressing high levels of the KGF receptors compared with cells expressing low levels of KGF receptors. Overall, this novel design may help with the targeting of specific cells that overexpress growth factor receptors such as KGF receptors.

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11 ± 0.04 h(-1)) is almost twice that of digestion without stretch (0.069 ± 0.028 h(-1)). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation.

Peripheral nerve regeneration can be enhanced by chemical and mechanical cues for neurite growth. Aligned and randomly oriented electrospun nanofibers of poly(ε-caprolactone) (PCL) or a blend of PCL and elastin were fabricated to test their potential to provide contact guidance to embryonic chick dorsal root ganglia for peripheral nerve regeneration. Scanning electron microscopy was used to analyze the fiber diameter. Fiber diameter was found to be significantly smaller when elastin was incorporated into the scaffold (934 ± 58 nm for PCL and 519 ± 36 nm for PCL:elastin). After 24 h in culture, there was preferential cell attachment and neurite extension along the fibers of the elastin-containing scaffolds (average neurite extension 173.4 ± 20.7 μm), indicating that the presence of elastin promotes neurite outgrowth on electrospun scaffolds.

Elastin is an extracellular matrix protein, providing elasticity to the organs, such as skin, blood vessels, lungs and elastic ligaments, presenting self-assembling ability to form elastic fibers. The elastin protein, as a component of elastin fibers, is one of the major proteins found in connective tissue and is responsible for the elasticity of tissues. It provides resilience to the human body, assembled as a continuous mesh of fibers that require to be deformed repetitively and reversibly. Thus, it is of great importance to investigate the development of the nanostructural surface of elastin-based biomaterials. The purpose of this research was to image the self-assembling process of elastin fiber structure under different experimental parameters such as suspension medium, elastin concentration, temperature of stock suspension and time interval after the preparation of the stock suspension. atomic force microscopy (AFM) was applied in order to investigate how different experimental parameters affected fiber development and morphology. The results demonstrated that through altering a number of experimental parameters, it was possible to affect the self-assembly procedure of elastin fibers from nanofibers and the formation of elastin nanostructured mesh consisting of naturally occurring fibers. Further clarification of the contribution of different parameters on fibril formation will enable the design and control of elastin-based nanobiomaterials with predetermined characteristics.

Elastin microfibril interface located protein 2 (EMILIN2) is an extracellular glycoprotein associated with cardiovascular development. While other EMILIN proteins are reported to play a role in elastogenesis and coagulation, little is known about EMILIN2 function in the cardiovascular system. The objective of this study was to determine whether EMILIN2 could play a role in thrombosis.

The development of adhesives that can be applied and create strong bonds underwater is a significant challenge for materials engineering. When the adhesive is intended for biomedical applications, further criteria, such as biocompatibility, must be met. Current biomedical adhesive technologies do not meet these needs. In response, we designed a bioinspired protein system that shows promise to achieve biocompatible underwater adhesion coupled with environmentally responsive behavior that is "smart" - that is, it can be tuned to suit a specific application. The material, ELY16, is constructed from an elastin-like polypeptide (ELP) that can be produced in high yields from Escherichia coli and can coacervate in response to environmental factors such as temperature, pH, and salinity. To confer wet adhesion, we utilized design principles from marine organisms such as mussels and sandcastle worms. When expressed, ELY16 is rich in tyrosine. Upon modification with the tyrosinase enzyme to form mELY16, the tyrosine residues are converted to 3,4-dihydroxyphenylalanine (DOPA). Both ELY16 and mELY16 exhibit cytocompatibility and significant dry adhesion strength (>2 MPa). Modification with DOPA increases protein adsorption to glass and provides moderate adhesion strength (∼240 kPa) in a highly humid environment. Furthermore, this ELP exhibits a tunable phase transition behavior that can be formulated to coacervate in physiological conditions and provides a convenient mechanism for application underwater. Finally, mELY16 possesses significantly higher adhesion strength in dry, humid, and underwater environments compared with a commercially available fibrin sealant. To our knowledge, mELY16 provides the strongest bonds of any rationally designed protein when used completely underwater, and its high yields make it more viable for commercial application compared to natural adhesive proteins. In conclusion, this ELP shows great potential to be a new "smart" underwater adhesive.