Targeted modulation of tropoelastin structure and assembly

Tropoelastin, as the monomer unit of elastin, assembles into elastic fibers that impart strength and resilience to elastic tissues. Tropoelastin is also widely used to manufacture versatile materials with specific mechanical and biological properties. The assembly of tropoelastin into elastic fibers or biomaterials is crucially influenced by key submolecular regions and specific residues within these domains. In this work, we identify the functional contributions of two rarely occurring negatively charged residues, glutamate 345 in domain 19 and glutamate 414 in domain 21, in jointly maintaining the native conformation of the tropoelastin hinge, bridge and foot regions. Alanine substitution of E345 and/or E414 variably alters the positioning and interactive accessibility of these regions, as illustrated by nanostructural studies and detected by antibody and cell probes. These structural changes are associated with a lower propensity for monomer coacervation, cross-linking into morphologically and functionally atypical hydrogels, and markedly impaired and abnormal elastic fiber formation. Our work indicates the crucial significance of both E345 and E414 residues in modulating specific local structure and higher-order assembly of human tropoelastin

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

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.United States. Office of Naval Research (Presidential Early Career Award for Scientists and Engineers)National Institutes of Health (U.S.) (U01 EB014976

Domains 12 to 16 of tropoelastin promote cell attachment and spreading through interactions with glycosaminoglycan and integrins alphaV and alpha5beta1

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 aV and a5b1 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 aV and a5b1 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

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

Stress-induced senescence in mesenchymal stem cells: Triggers, hallmarks, and current rejuvenation approaches

Mesenchymal stem cells (MSCs) have emerged as promising cell-based therapies in the treatment of degenerative and inflammatory conditions. However, despite accumulating evidence of the breadth of MSC functional potency, their broad clinical translation is hampered by inconsistencies in therapeutic efficacy, which is at least partly due to the phenotypic and functional heterogeneity of MSC populations as they progress towards senescence in vitro. MSC senescence, a natural response to aging and stress, gives rise to altered cellular responses and functional decline. This review describes the key regenerative properties of MSCs; summarises the main triggers, mechanisms, and consequences of MSC senescence; and discusses current cellular and extracellular strategies to delay the onset or progression of senescence, or to rejuvenate biological functions lost to senescence

Molecular Model of Human Tropoelastin and Implications of Associated Mutations

Protein folding poses unique challenges for large, disordered proteins due to the low resolution of structural data accessible in experiment and on the basis of short time scales and limited sampling attainable in computation. Such molecules are uniquely suited to accelerated-sampling molecular dynamics algorithms due to a flat-energy landscape. We apply these methods to report here the folded structure in water from a fully extended chain of tropoelastin, a 698-amino acid molecular precursor to elastic fibers that confer elasticity and recoil to tissues, finding good agreement with experimental data. We then study a series of artificial and disease-related mutations, yielding molecular mechanisms to explain structural differences and variation in hierarchical assembly observed in experiment. The present model builds a framework for studying assembly and disease and yields critical insight into molecular mechanisms behind these processes. These results suggest that proteins with disordered regions are suitable candidates for characterization by this approach. Keywords: tropoelastin; elastic fiber; structural protein; disordered protein; molecular dynamicsNational Institutes of Health (U.S.) (Grant U01 HS 4976)United States. Office of Naval Research (Grant N00014-16-1-2333

Tropoelastin is a Flexible Molecule that Retains its Canonical Shape

Tropoelastin is the dominant building block of elastic fibers, which form a major component of the extracellular matrix, providing structural support to tissues and imbuing them with elasticity and resilience. Recently, the atomistic structure of human tropoelastin is described, obtained through accelerated sampling via replica exchange molecular dynamics simulations. Here, principal component analysis is used to consider the ensemble of structures accessible to tropoelastin at body temperature (37 °C) at which tropoelastin naturally self-assembles into aggregated coacervates. These coacervates are relevant because they are an essential intermediate assembly stage, where tropoelastin molecules are then cross-linked at lysine residues and integrated into growing elastic fibers. It is found that the ensemble preserves the canonical tropoelastin structure with an extended molecular body flanked by two protruding legs, and identifies variations in specific domain positioning within this global shape. Furthermore, it is found that lysine residues show a large variation in their location on the tropoelastin molecule compared with other residues. It is hypothesized that this perturbation of the lysines increases their accessibility and enhances cross-linking. Finally, the principal component modes are extracted to describe the range of tropoelastin's conformational fluctuation to validate tropoelastin's scissor-twist motion that was predicted earlier.NIH (Grant U01HS4976)Office of Naval Research (Grant N00014‐16‐1‐2333

Extracellular Vesicles: Interplay with the Extracellular Matrix and Modulated Cell Responses

The discovery that cells secrete extracellular vesicles (EVs), which carry a variety of regulatory proteins, nucleic acids, and lipids, has shed light on the sophisticated manner by which cells can communicate and accordingly function. The bioactivity of EVs is not only defined by their internal content, but also through their surface associated molecules, and the linked downstream signaling effects they elicit in target cells. The extracellular matrix (ECM) contains signaling and structural molecules that are central to tissue maintenance and repair. Recently, a subset of EVs residing within the extracellular matrix has been identified. Although some roles have been proposed for matrix-bound vesicles, their role as signaling molecules within the ECM is yet to be explored. Given the close association of EVs and the ECM, it is not surprising that EVs partly mediate repair and regeneration by modulating matrix deposition and degradation through their cellular targets. This review addresses unique EV features that allow them to interact with and navigate through the ECM, describes how their release and content is influenced by the ECM, and emphasizes the emerging role of stem-cell derived EVs in tissue repair and regeneration through their matrix-modulating properties

Plasma ion implantation enabled bio-functionalization of PEEK improves osteoblastic activity

Slow appositional growth of bone in vivo is a major problem associated with polyether ether ketone (PEEK) based orthopaedic implants. Early stage promotion of osteoblast activity, particularly bone nodule formation, would help to improve contact between PEEK implantable materials and the surrounding bone tissue. To improve interactions with bone cells, we explored here the use of plasma immersion ion implantation (PIII) treatment of PEEK to covalently immobilize biomolecules to the surface. In this study, a single step process was used to covalently immobilize tropoelastin on the surface of PIII modified PEEK through reactions with radicals generated by the treatment. Improved bioactivity was observed using the human osteoblast-like cell line, SAOS-2. Cells on surfaces that were PIII-treated or tropoelastin-coated exhibited improved attachment, spreading, proliferation, and bone nodule formation compared to cells on untreated samples. Surfaces that were both PIII-treated and tropoelastin-coated triggered the most favorable osteoblast-like responses. Surface treatment or tropoelastin coating did not alter alkaline phosphatase gene expression and activity of bound cells but did influence the expression of other bone markers including osteocalcin, osteonectin, and collagen I. We conclude that the surface modification of PEEK improves osteoblast interactions, particularly with respect to bone apposition, and enhances the orthopedic utility of PEEK