Giselle C. Yeo

Coacervation of tropoelastin

The coacervation of tropoelastin represents the first major stage of elastic fiber assembly. The process has been modeled in vitro by numerous studies, initially with mixtures of solubilized elastin, and subsequently with synthetic elastin peptides that represent hydrophobic repeat units, isolated hydrophobic domains, segments of alternating hydrophobic and cross-linking domains, or the full-length monomer. Tropoelastin coacervation in vitro is characterized by two stages: an initial phase separation, which involves a reversible inverse temperature transition of monomer to n-mer; and maturation, which is defined by the irreversible coalescence of coacervates into large species with fibrillar structures. Coacervation is an intrinsic ability of tropoelastin. It is primarily influenced by the number, sequence, and contextual arrangement of hydrophobic domains, although hydrophilic sequences can also affect the behavior of the hydrophobic domains and thus affect coacervation. External conditions including ionic strength, pH, and temperature also directly influence the propensity of tropoelastin to self-associate. Coacervation is an endothermic, entropically-driven process driven by the cooperative interactions of hydrophobic domains following destabilization of the clathrate-like water shielding these regions. The formation of such assemblies is believed to follow a helical nucleation model of polymerization. Coacervation is closely associated with conformational transitions of the monomer, such as increased β-structures in hydrophobic domains and α-helices in cross-linking domains. Tropoelastin coacervation in vivo is thought to mainly involve the central hydrophobic domains. In addition, cell-surface glycosaminoglycans and microfibrillar proteins may regulate the process. Coacervation is essential for progression to downstream elastogenic stages, and impairment of the process can result in elastin haploinsufficiency disorders such as supravalvular aortic stenosis.

Tropoelastin - a versatile, bioactive assembly module

Elastin provides structural integrity, biological cues and persistent elasticity to a range of important tissues, including the vasculature and lungs. Its critical importance to normal physiology makes it a desirable component of biomaterials that seek to repair or replace these tissues. The recent availability of large quantities of the highly purified elastin monomer, tropoelastin, has allowed for a thorough characterization of the mechanical and biological mechanisms underpinning the benefits of mature elastin. While tropoelastin is a flexible molecule, a combination of optical and structural analyses has defined key regions of the molecule that directly contribute to the elastomeric properties and control the cell interactions of the protein. Insights into the structure and behavior of tropoelastin have translated into increasingly sophisticated elastin-like biomaterials, evolving from classically manufactured hydrogels and fibers to new forms, stabilized in the absence of incorporated cross-linkers. Tropoelastin is also compatible with synthetic and natural co-polymers, expanding the applications of its potential use beyond traditional elastin-rich tissues and facilitating finer control of biomaterial properties and the design of next-generation tailored bioactive materials.

Tropoelastin bridge region positions the cell-interactive C terminus and contributes to elastic fiber assembly

The tropoelastin monomer undergoes stages of association by coacervation, deposition onto microfibrils, and cross-linking to form elastic fibers. Tropoelastin consists of an elastic N-terminal coil region and a cell-interactive C-terminal foot region linked together by a highly exposed bridge region. The bridge region is conveniently positioned to modulate elastic fiber assembly through association by coacervation and its proximity to dominant cross-linking domains. Tropoelastin constructs that either modify or remove the entire bridge and downstream regions were assessed for elastogenesis. These constructs focused on a single alanine substitution (R515A) and a truncation (M155n) at the highly conserved arginine 515 site that borders the bridge. Each form displayed less efficient coacervation, impaired hydrogel formation, and decreased dermal fibroblast attachment compared to wild-type tropoelastin. The R515A mutant protein additionally showed reduced elastic fiber formation upon addition to human retinal pigmented epithelium cells and dermal fibroblasts. The small-angle X-ray scattering nanostructure of the R515A mutant protein revealed greater conformational flexibility around the bridge and C-terminal regions. This increased flexibility of the R515A mutant suggests that the tropoelastin R515 residue stabilizes the structure of the bridge region, which is critical for elastic fiber assembly.

Mechanical properties of plasma immersion ion implanted PEEK for bioactivation of medical devices

Plasma immersion ion implantation (PIII) is used to modify the surface properties of polyether ether ketone for biomedical applications. Modifications to the mechanical and chemical properties are characterized as a function of ion fluence (treatment time) to determine the suitability of the treated surfaces for biological applications. Youngs modulus and elastic recovery were found to increase with respect to treatment time at the surface from 4.4 to 5.2 MPa and from 0.49 to 0.68, respectively. The mechanical properties varied continuously with depth, forming a graded layer where the mechanical properties returned to untreated values deep within the layer. The treated surface layer exhibited cracking under cyclical loads, associated with an increased modulus due to dehydrogenation and cross-linking; however, it did not show any sign of delamination, indicating that the modified layer is well integrated with the substrate, a critical factor for bioactive surface coatings. The oxygen concentration remained unchanged at the surface; however, in contrast to ion implanted polymers containing only carbon and hydrogen, the oxygen concentration within the treated layer was found to decrease. This effect is attributed to UV exposure and suggests that PIII treatments can modify the surface to far greater depths than previously reported. Protein immobilization on PIII treated surfaces was found to be independent of treatment time, indicating that the surface mechanical properties can be tuned for specific applications without affecting the protein coverage. Our findings on the mechanical properties demonstrate such treatments render PEEK well suited for use in orthopedic implantable devices.

Tropoelastin coated PLLA-PLGA scaffolds promote vascular network formation.

The robust repair of large wounds and tissue defects relies on blood flow. This vascularization is the major challenge faced by tissue engineering on the path to forming thick, implantable tissue constructs. Without this vasculature, oxygen and nutrients cannot reach the cells located far from host blood vessels. To make viable constructs, tissue engineering takes advantage of the mechanical properties of synthetic materials, while combining them with ECM proteins to create a natural environment for the tissue-specific cells. Tropoelastin, the precursor of the elastin, is the ECM protein responsible for elasticity in diverse tissues, including robust blood vessels. Here, we seeded endothelial cells with supporting cells on PLLA/PLGA scaffolds treated with tropoelastin, and examined the morphology, expansion and maturity of the newly formed vessels. Our results demonstrate that the treated scaffolds elicit a more expanded, complex and developed vascularization in comparison to the untreated group. Implantation of tropoelastin-treated scaffolds into mouse abdominal muscle resulted in enhanced perfusion of the penetrating vasculature and improved integration. This study points to the great potential of these combined materials in promoting the vascularization of implanted engineered constructs, which can be further exploited in the fabrication of clinically relevant engineered tissues.

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

ABSTRACTPurposeTo 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.MethodsRecombinant human TE and a point mutant variant (R515A) of TE were labeled with 125Iodine 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.ResultsTE 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.ConclusionsProtein 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.

Subtle balance of tropoelastin molecular shape and flexibility regulates dynamics and hierarchical assembly

Tropoelastin’s local and global structures dictate molecular dynamics and are essential for efficient assembly into elastin. The assembly of the tropoelastin monomer into elastin is vital for conferring elasticity on blood vessels, skin, and lungs. Tropoelastin has dual needs for flexibility and structure in self-assembly. We explore the structure-dynamics-function interplay, consider the duality of molecular order and disorder, and identify equally significant functional contributions by local and global structures. To study these organizational stratifications, we perturb a key hinge region by expressing an exon that is universally spliced out in human tropoelastins. We find a herniated nanostructure with a displaced C terminus and explain by molecular modeling that flexible helices are replaced with substantial β sheets. We see atypical higher-order cross-linking and inefficient assembly into discontinuous, thick elastic fibers. We explain this dysfunction by correlating local and global structural effects with changes in the molecule’s assembly dynamics. This work has general implications for our understanding of elastomeric proteins, which balance disordered regions with defined structural modules at multiple scales for functional assembly.

A Negatively Charged Residue Stabilizes the Tropoelastin N-terminal Region for Elastic Fiber Assembly

Background: Negative residues are rare in human tropoelastin, and their contributions to protein structure and function are unknown. Results: Mutating aspartate 72 reduces coacervation, cross-linking, and elastogenesis and alters the N-terminal conformation. Conclusion: Aspartate 72 residue stabilizes the N-terminal structure for functional assembly into elastic fibers. Significance: This work provides the first direct evidence of the importance of the tropoelastin N-terminal structure in elastic fiber assembly. Tropoelastin is an extracellular matrix protein that assembles into elastic fibers that provide elasticity and strength to vertebrate tissues. Although the contributions of specific tropoelastin regions during each stage of elastogenesis are still not fully understood, studies predominantly recognize the central hinge/bridge and C-terminal foot as the major participants in tropoelastin assembly, with a number of interactions mediated by the abundant positively charged residues within these regions. However, much less is known about the importance of the rarely occurring negatively charged residues and the N-terminal coil region in tropoelastin assembly. The sole negatively charged residue in the first half of human tropoelastin is aspartate 72. In contrast, the same region comprises 17 positively charged residues. We mutated this aspartate residue to alanine and assessed the elastogenic capacity of this novel construct. We found that D72A tropoelastin has a decreased propensity for initial self-association, and it cross-links aberrantly into denser, less porous hydrogels with reduced swelling properties. Although the mutant can bind cells normally, it does not form elastic fibers with human dermal fibroblasts and forms fewer atypical fibers with human retinal pigmented epithelial cells. This impaired functionality is associated with conformational changes in the N-terminal region. Our results strongly point to the role of the Asp-72 site in stabilizing the N-terminal segment of human tropoelastin and the importance of this region in facilitating elastic fiber assembly.

Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin

The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.

A sterilizable, biocompatible, tropoelastin surface coating immobilized by energetic ion activation

Biomimetic materials which integrate with surrounding tissues and regulate new tissue formation are attractive for tissue engineering and regenerative medicine. Plasma immersion ion-implanted (PIII) polyethersulfone (PES) provides an excellent platform for the irreversible immobilization of bioactive proteins and peptides. PIII treatment significantly improves PES wettability and results in the formation of acidic groups on the PES surface, with the highest concentration observed at 40–80 s of PIII treatment. The elastomeric protein tropoelastin can be stably adhered to PIII-treated PES in a cell-interactive conformation by tailoring the pH and salt levels of the protein–surface association conditions. Tropoelastin-coated PIII-treated PES surfaces are resistant to molecular fouling, and actively promote high levels of fibroblast adhesion and proliferation while maintaining cell morphology. Tropoelastin, unlike other extracellular matrix proteins such as fibronectin, uniquely retains full bioactivity even after medical-grade ethylene oxide sterilization. This dual approach of PIII treatment and tropoelastin cloaking allows for the stable, robust functionalization of clinically used polymer materials for directed cellular interactions.