%0 Journal Article %~ PubMed %A Nivison-Smith, Lisa %A Weiss, Anthony S %T Alignment of human vascular smooth muscle cells on parallel electrospun synthetic elastin fibers. %B Journal of biomedical materials research. Part A %D 2012 %C United States %I John Wiley & Sons, Inc. %V 100 %N 1 %P 155-61 %@ 1552-4965 %X 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 ??-smooth muscle actin. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Bax, Daniel V %A Tipa, Roxana S %A Kondyurin, Alexey %A Higgins, Michael J %A Tsoutas, Kostadinos %A Gelmi, Amy %A Wallace, Gordon G %A McKenzie, David R %A Weiss, Anthony S %A Bilek, Marcela M M %T Cell patterning via linker-free protein functionalization of an organic conducting polymer (polypyrrole) electrode. %B Acta Biomaterialia %D 2012 %C Netherlands %I Elsevier BV %V 8 %N 7 %P 2538-2548 %@ 1878-7568 %X The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of the conducting polymer, polypyrrole, which possesses electrical properties. PIII treatment enabled persistent, covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. In contrast tropoelastin was readily eluted from the untreated surface. Through this differential persistence of binding, surface bound tropoelastin supported cell adhesion and spreading on the PIII treated but not the untreated polypyrrole surface. The application of a steel shadow mask during PIII treatment allowed for spatial definition of tropoelastin exclusively to PIII treated regions. The general applicability of this approach to other extracellular matrix proteins was illustrated using collagen I, which displayed similar results to tropoelastin but required extended washing conditions. This approach allowed fine patterning of cell adhesion and spreading to tropoelastin and collagen, specifically on PIII treated polypyrrole regions. We therefore present a methodology to alter the functionality of polypyrrole surfaces, generating surfaces that can spatially control cellular interactions through protein functionalization with the potential for electrical stimulation. %Z FOR Codes: 90301 %0 Journal Article %~ PubMed %A Reddel, Caroline J %A Weiss, Anthony S %A Burgess, Janette K %T Elastin in asthma. %B Pulmonary Pharmacology & Therapeutics %D 2012 %C United Kingdom %I Academic Press %V 25 %N 2 %P 144-153 %@ 1522-9629 %X Extracellular matrix is generally increased in asthma, causing thickening of the airways which may either increase or decrease airway responsiveness, depending on the mechanical requirements of the deposited matrix. However, in??vitro studies have shown that the altered extracellular matrix produced by asthmatic airway smooth muscle cells is able to induce increased proliferation of non-asthmatic smooth muscle cells, which is a process believed to contribute to airway hyper-responsiveness in asthma. Elastin is an extracellular matrix protein that is altered in asthmatic airways, but there has been no systematic investigation of the functional effect of these changes. This review reveals divergent reports of the state of elastin in the airway wall in asthma. In some layers of the airway it has been described as increased, decreased and/or fragmented, or unchanged. There is also considerable evidence for an imbalance of matrix metalloproteinases, which degrade elastin, and their respective inhibitors the tissue inhibitors of metalloproteinases, which collectively help to explain observations of both increased elastin and elastin fragments. A loss of lung elastic recoil in asthma suggests a mechanical role for disordered elastin in the aetiology of the disease, but extensive studies of elastin in other tissues show that elastin fragments elicit cellular effects such as increased proliferation and inflammation. This review summarises the current understanding of the role of elastin in the asthmatic airway. %Z FOR Codes: 111501 %0 Journal Article %~ PubMed %A Almine, Jessica F %A Wise, Steven G %A Weiss, Anthony S %T Elastin signaling in wound repair. %B Birth Defects Research. Part C: Embryo Today Reviews %D 2012 %C United States %I John Wiley & Sons, Inc. %V 96 %N 3 %P 248-257 %@ 1542-9768 %X %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Zhao, Hui-Lin %A Chen, Xiu-Lan %A Xie, Bin-Bin %A Zhou, Ming-Yang %A Gao, Xiang %A Zhang, Xi-Ying %A Zhou, Bai-Cheng %A Weiss, Anthony S %A Zhang, Yu-Zhong %T Elastolytic mechanism of a novel M23 metalloprotease pseudoalterin from deep-sea Pseudoalteromonas sp. CF6-2: cleaving not only glycyl bonds in the hydrophobic regions, but also peptide bonds in the hydrophilic regions involved in crosslinking. %B Journal of Biological Chemistry %D 2012 %C United States %I American Society for Biochemistry and Molecular B %V 287 %N 47 %P 39710-39720 %@ 0021-9258 %X %Z FOR Codes: 60104 %0 Journal Article %~ PubMed %A Rnjak-Kovacina, Jelena %A Wise, Steven G %A Li, Zhe %A Maitz, Peter K M %A Young, Cara J %A Wang, Yiwei %A Weiss, Anthony S %T Electrospun synthetic human elastin:collagen composite scaffolds for dermal tissue engineering. %B Acta Biomaterialia %D 2012 %C Netherlands %I Elsevier BV %V 8 %N 10 %P 3714-3722 %@ 1878-7568 %X We present 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 6 weeks. The 80T20C scaffolds supported fibroblast infiltration, de novo collagen deposition and new capillary formation. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Le Brun, Anton P %A Chow, John %A Bax, Daniel V %A Nelson, Andrew %A Weiss, Anthony S %A James, Michael %T Molecular orientation of tropoelastin is determined by surface hydrophobicity. %B Biomacromolecules %D 2012 %C United States %I American Chemical Society %V 13 %N 2 %P 379-386 %@ 1526-4602 %X %Z FOR Codes: 60106 60109 %0 Journal Article %~ PubMed %A Wise, Steven G %A Waterhouse, Anna %A Kondyurin, Alexey %A Bilek, Marcela M %A Weiss, Anthony S %T Plasma-based biofunctionalization of vascular implants. %B Nanomedicine %D 2012 %C United Kingdom %I Future Medicine Ltd. %V 7 %N 12 %P 1907-1916 %@ 1748-6963 %X %Z FOR Codes: 29901 %0 Journal Article %A Hu, Xiao %A Cebe, Peggy %A Weiss, Anthony %A Omenetto, Fiorenzo %A Kaplan, David L. %T Protein-based composite materials %B Materials Today %D 2012 %C Netherlands %I Elsevier BV %V 15 %N 5 %P 208-215 %@ 1369-7021 %X %Z FOR Codes: 30406 %0 Journal Article %~ PubMed %A Ohgo, Kosuke %A Niemczura, Walter P %A Seacat, Brian C %A Wise, Steven G %A Weiss, Anthony S %A Kumashiro, Kristin K %T Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy. %B Journal of Biological Chemistry %D 2012 %C United States %I American Society for Biochemistry and Molecular Bi %V 287 %N 22 %P 18201-18209 %@ 1083-351X %X 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. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Heinz, Andrea %A Jung, Michael C %A Jahreis, Günther %A Rusciani, Anthony %A Duca, Laurent %A Debelle, Laurent %A Weiss, Anthony S %A Neubert, Reinhard H H %A Schmelzer, Christian E H %T The action of neutrophil serine proteases on elastin and its precursor. %B Biochimie %D 2012 %C France %I Elsevier Masson %V 94 %N 1 %P 192-202 %@ 1638-6183 %X This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P(1) in tropoelastin, whereas Lys was not identified at P(1) in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P(2) and P(4)''. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Yeo, Giselle C %A Baldock, Clair %A Tuukkanen, Anne %A Roessle, Manfred %A Dyksterhuis, Leanne B %A Wise, Steven G %A Matthews, Jacqueline %A Mithieux, Suzanne M %A Weiss, Anthony S %T Tropoelastin bridge region positions the cell-interactive C terminus and contributes to elastic fiber assembly. %B Proceedings of the National Academy of Sciences of the United States of America %D 2012 %C United States %I National Academy of Sciences %V 109 %N 8 %P 2878-2883 %@ 0027-8424 %X 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. %Z FOR Codes: 60112 %0 Journal Article %~ PubMed %A Wise, Steven G %A Byrom, Michael J %A Waterhouse, Anna %A Bannon, Paul G %A Ng, Martin K C %A Weiss, Anthony S %T A multilayered synthetic human elastin/polycaprolactone hybrid vascular graft with tailored mechanical properties. %B Acta biomaterialia %D 2011 %C Netherlands %I Elsevier BV %V 7 %N 1 %P 295-303 %@ 1878-7568 %X Small-diameter synthetic vascular graft materials fail to match the patency of human tissue conduits used in vascular bypass surgery. The foreign surface retards endothelialization and is highly thrombogenic, while the mismatch in mechanical properties induces intimal hyperplasia. Using recombinant human tropoelastin, we have developed a synthetic vascular conduit for small-diameter applications. We show that tropoelastin enhances endothelial cell attachment (threefold vs. control) and proliferation by 54.7 ?? 1.1% (3 days vs. control). Tropoelastin, when presented as a monomer and when cross-linked into synthetic elastin for biomaterials applications, had low thrombogenicity. Activation of the intrinsic pathway of coagulation, measured by plasma clotting time, was reduced for tropoelastin (60.4 ?? 8.2% vs. control). Platelet attachment was also reduced compared to collagen. Reductions in platelet interactions were mirrored on cross-linked synthetic elastin scaffolds. Tropoelastin was subsequently incorporated into a synthetic elastin/polycaprolactone conduit with mechanical properties optimized to mimic the human internal mammary artery, including permeability, compliance, elastic modulus and burst pressure. Further, this multilayered conduit presented a synthetic elastin internal lamina to circulating blood and demonstrated suturability and mechanical durability in a small scale rabbit carotid interposition model. %Z FOR Codes: 100404 %0 Journal Article %~ PubMed %A Bax, Daniel V %A Wang, Yiwei %A Li, Zhe %A Maitz, Peter K M %A McKenzie, David R %A Bilek, Marcela M M %A Weiss, Anthony S %T Binding of the cell adhesive protein tropoelastin to PTFE through plasma immersion ion implantation treatment. %B Biomaterials %D 2011 %C Netherlands %I Elsevier BV %V 32 %N 22 %P 5100-11 %@ 0142-9612 %X The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of polytetrafluorethylene (PTFE), enabling the covalent binding of a cell adhesive protein, tropoelastin, without employing chemical linking molecules. Tropoelastin coating of untreated or PIII treated PFTE simultaneously promoted and blocked cell interactions respectively, i.e. PIII treatment of the PTFE surface completely inverses the cell interactive properties of bound tropoelastin. This activity persisted over long term storage of the PIII treated surfaces. The integrin binding C-terminus of tropoelastin was markedly less solvent exposed when bound to PIII treated PTFE than untreated PTFE, accounting for the modulation of cell adhesive activity. This presents a new methodology to specifically modulate cell behavior on a polymer surface using a simple one step treatment process, by adjusting the adhesive activity of a single extracellular matrix protein. %Z FOR Codes: 903 %0 Journal Article %~ PubMed %A Yeo, Giselle C %A Keeley, Fred W %A Weiss, Anthony S %T Coacervation of tropoelastin. %B Advances in colloid and interface science %D 2011 %C Netherlands %I Elsevier BV %V 167 %N 1-2 %P 94-103 %@ 1873-3727 %X 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 ??-structures in hydrophobic domains and ??-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. %Z FOR Codes: 30406 %0 Journal Article %~ PubMed %A Hayes, Anthony J %A Lord, Megan S %A Smith, Susan M %A Smith, Margaret M %A Whitelock, John M %A Weiss, Anthony S %A Melrose, James %T Colocalization in vivo and association in vitro of perlecan and elastin. %B Histochemistry and Cell Biology %D 2011 %C Germany %I Springer %V 136 %N 4 %P 437-454 %@ 1432-119X %X We have colocalized elastin and fibrillin-1 with perlecan in extracellular matrix of tensional and weight-bearing connective tissues. Elastin and fibrillin-1 were identified as prominent components of paraspinal blood vessels, and posterior longitudinal ligament in the human fetal spine and outer annulus fibrosus of the fetal intervertebral disc. We also colocalized perlecan with a synovial elastic basal lamina, where the attached synovial cells were observed to produce perlecan. Elastin, fibrillin-1 and perlecan were co-localized in the intima and media of small blood vessels in the synovium and in human fetal paraspinal blood vessels. Elastic fibers were observed at the insertion point of the anterior cruciate ligament to bone in the ovine stifle joint where they colocalized with perlecan. Elastin has not previously been reported to be spatially associated with perlecan in these tissues. Interactions between the tropoelastin and perlecan heparan sulfate chains were demonstrated using quartz crystal microbalance with dissipation solid phase binding studies. Electrostatic interactions through the heparan sulfate chains of perlecan and core protein mediated the interactions with tropoelastin, and were both important in the coacervation of tropoelastin and deposition of elastin onto perlecan immobilized on the chip surface. This may help us to understand the interactions which are expected to occur in vivo between the tropoelastin and perlecan to facilitate the deposition of elastin and formation of elastic microfibrils in situ and would be consistent with the observed distributions of these components in a number of connective tissues. %Z FOR Codes: 60112 %0 Journal Article %~ PubMed %A Bax, Daniel V %A McKenzie, David R %A Bilek, Marcela M M %A Weiss, Anthony S %T Directed cell attachment by tropoelastin on masked plasma immersion ion implantation treated PTFE. %B Biomaterials %D 2011 %C Netherlands %I Elsevier BV %V 32 %N 28 %P 6710-8 %@ 0142-9612 %X The ability to generate cell patterns on polymer surfaces is critical for the detailed study of cellular biology, the fabrication of cell-based biosensors, cell separation techniques and for tissue engineering. In this study contact tape masking and steel shadow masks were used to exclude plasma immersion ion implantation (PIII) treatment from defined areas of polytetrafluoroethylene (PTFE) surfaces. This process enabled patterned covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. Tropoelastin coating rendered the untreated regions cell adhesive and the PIII-treated area non-adhesive, allowing very fine patterning of cell adhesion to PTFE surfaces. A blocking step, such as with BSA or PEG, was not required to prevent cell binding to the underlying PIII-treated regions as tropoelastin coating alone performed this blocking function. Although tropoelastin coated the entire PTFE surface, the cell binding C-terminus of tropoelastin was markedly less solvent exposed on the PIII-treated, hydrophilic regions. The differential exposure of the C-terminus correlated with the patterned distribution of tropoelastin-mediated cell adhesion. This new methodology specifically enables directed cell behavior on a polymer surface using a simple one-step treatment process, by modulating the adhesive activity of a single extracellular matrix protein. %Z FOR Codes: 60106 %0 Book Section %A Nivison-Smith, Lisa %A Weiss, Anthony %T Elastin Based Constructs %B Regenerative Medicine and Tissue Engineering- Cells and Biomaterials %D 2011 %C Croatia %I InTech %V %N %P 323-340 %@ 9789533076638 %E Eberli, Daniel %X %Z FOR Codes: 100404 %0 Journal Article %~ PubMed %A Waterhouse, Anna %A Wise, Steven G %A Ng, Martin K C %A Weiss, Anthony S %T Elastin as a nonthrombogenic biomaterial. %B Tissue engineering. Part B, Reviews %D 2011 %C United States %I Mary Ann Liebert, Inc. Publishers %V 17 %N 2 %P 93-9 %@ 1937-3376 %X Surface-induced thrombosis is a significant issue for artificial blood-contacting materials used in the treatment of cardiovascular diseases. The development of biomaterials and tissue-engineered constructs that mimic the vasculature represents a way to overcome this problem. Elastin is an extracellular matrix macromolecule that imparts arterial elasticity where it comprises up to 50% of the nonhydrated mass of the vessel. In addition to its critical role in maintaining vessel integrity and elastic properties under pulsatile flow, elastin plays an important role in signaling and regulating luminal endothelial cells and smooth muscle cells in the arterial wall. Despite its well-established significance in the vasculature and its growing use as a biomaterial in tissue engineering, the hemocompatibility of elastin is often overlooked. Past studies pointing to the potential of arterial elastin and decellularized elastin as nonthrombogenic materials have begun to be realized, with elastin scaffolds and coatings displaying increased hemocomptibility. This review explores the mechanisms of elastin''s nonthrombogenicity and highlights the current problems limiting its wider application as a biomaterial. We discuss the benefits of constructing biomaterials encompassing the relevant mechanical and biological features of elastin to provide enhanced hemocompatibility to biomaterials. %Z FOR Codes: 60106 %0 Book Section %A Rnjak-Kovacina, J. %A Daamen, W.F. %A Pierna, M %A Rodríguez-Cabello, J.C %A Weiss, Anthony %T Elastin biopolymers %B Comprehensive Biomaterials %D 2011 %C United Kingdom %I Elsevier Science Ltd %V %N %P 329-346 %@ 9780080553023 %E Kirkpatrick, C. James %E Grainger, David W. %E Hutmacher, Dietmar Werner %E Healy, Kevin E. %E Ducheyne, Paul %X %Z FOR Codes: 100404 %0 Journal Article %A Annabi, Nasim %A Ali Fathi, Ali %A Mithieux, Suzanne %A Weiss, Anthony %A Dehghani, Fariba %T Fabrication of porous PCL/elastin composite scaffolds for tissue engineering applications %B Journal of Supercritical Fluids %D 2011 %C Netherlands %I Elsevier BV %V 59 %N %P 157-167 %@ 0896-8446 %X %Z FOR Codes: 90399 %0 Journal Article %~ PubMed %A Bilek, Marcela M M %A Bax, Daniel V %A Kondyurin, Alexey %A Yin, Yongbai %A Nosworthy, Neil J %A Fisher, Keith %A Waterhouse, Anna %A Weiss, Anthony S %A Dos Remedios, Cristobal G %A McKenzie, David R %T Free radical functionalization of surfaces to prevent adverse responses to biomedical devices. %B Proceedings of the National Academy of Sciences of the United States of America %D 2011 %C United States %I National Academy of Sciences %V 108 %N 35 %P 14405-10 %@ 0027-8424 %X Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines. %Z FOR Codes: 30401 60106 %0 Journal Article %~ PubMed %A Rnjak-Kovacina, Jelena %A Weiss, Anthony S %T Increasing the Pore Size of Electrospun Scaffolds. %B Tissue engineering. Part B, Reviews %D 2011 %C United States %I Mary Ann Liebert, Inc. Publishers %V 17 %N 5 %P 365-72 %@ 1937-3376 %X Electrospinning has gained much attention in the past decade as an effective means of generating nano- to micro-scale polymer fibers that resemble native extracellular matrix. High porosity, pore interconnectivity, and large surface area to volume ratio of electrospun scaffolds make them highly conducive to cellular adhesion and growth. However, inherently small pores of electrospun scaffolds do not promote adequate cellular infiltration and tissue ingrowth. Cellular infiltration into the scaffold is essential for a range of tissue engineering applications and is particularly important in skin and musculoskeletal engineering. Pore size, porosity, and pore interconnectivity dictate the extent of cellular infiltration and tissue ingrowth into the scaffold; influence a range of cellular processes; and are crucial for diffusion of nutrients, metabolites, and waste products. A number of electrospinning techniques and postelectrospinning modifications have, therefore, been developed in order to increase the pore size of electrospun scaffolds. Diverse techniques ranging from simple variations in the electrospinning parameters to complex methodologies requiring highly specialized equipment have been explored and are described in this article. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Rnjak, Jelena %A Wise, Steven G %A Mithieux, Suzanne M %A Weiss, Anthony S %T Severe burn injuries and the role of elastin in the design of dermal substitutes. %B Tissue engineering. Part B, Reviews %D 2011 %C United States %I Mary Ann Liebert, Inc. Publishers %V 17 %N 2 %P 81-91 %@ 1937-3376 %X Severe burn injuries are a major health problem as they can compromise whole body function and result in extensive emotional trauma exacerbated by prolonged hospital stay. Burn injury treatment has improved dramatically to increase the probability of survival, but burn survivors still suffer from excessive scarring and skin contractures, which substantially compromise their health and quality of life. Elastin is historically underrepresented in commercial dermal substitutes, yet deserves consideration because of its fundamental role in skin structure and function. Dermal elastic network is a strong determinant of skin resilience, texture, and quality but is not sufficiently regenerated following burn injury. In addition to its structural and mechanical roles, elastin has inherent cell signaling properties that promote a diverse range of cellular responses including chemotaxis, cell attachment, proliferation, and differentiation. Scaffold elasticity and regeneration of the elastic fiber system is now recognized as integral to the development of functional dermal substitutes. Dermal substitutes are intended to replace damaged dermal tissue in severe burn injuries. Elastin-based dermal substitutes have the potential to decrease wound contraction, improve scar appearance and functionality, and contribute to wound healing outcomes through a combination of elastin''s mechanical and cell signaling properties. %Z FOR Codes: 100404 %0 Journal Article %~ PubMed %A Baldock, Clair %A Oberhauser, Andres F %A Ma, Liang %A Lammie, Donna %A Siegler, Veronique %A Mithieux, Suzanne M %A Tu, Yidong %A Chow, John Yuen Ho %A Suleman, Farhana %A Malfois, Marc %A Rogers, Sarah %A Guo, Liang %A Irving, Thomas C %A Wess, Tim J %A Weiss, Anthony S %T Shape of tropoelastin, the highly extensible protein that controls human tissue elasticity. %B Proceedings of the National Academy of Sciences of the United States of America %D 2011 %C United States %I National Academy of Sciences %V 108 %N 11 %P 4322-7 %@ 0027-8424 %X Elastin enables the reversible deformation of elastic tissues and can withstand decades of repetitive forces. Tropoelastin is the soluble precursor to elastin, the main elastic protein found in mammals. Little is known of the shape and mechanism of assembly of tropoelastin as its unique composition and propensity to self-associate has hampered structural studies. In this study, we solve the nanostructure of full-length and corresponding overlapping fragments of tropoelastin using small angle X-ray and neutron scattering, allowing us to identify discrete regions of the molecule. Tropoelastin is an asymmetric coil, with a protruding foot that encompasses the C-terminal cell interaction motif. We show that individual tropoelastin molecules are highly extensible yet elastic without hysteresis to perform as highly efficient molecular nanosprings. Our findings shed light on how biology uses this single protein to build durable elastic structures that allow for cell attachment to an appended foot. We present a unique model for head-to-tail assembly which allows for the propagation of the molecule''s asymmetric coil through a stacked spring design. %Z FOR Codes: 30406 %0 Journal Article %~ PubMed %A McGrath, Aaron P %A Mithieux, Suzanne M %A Collyer, Charles A %A Bakhuis, Janny G %A van den Berg, Marco %A Sein, Arjen %A Heinz, Andrea %A Schmelzer, Christian %A Weiss, Anthony S %A Guss, J Mitchell %T Structure and Activity of Aspergillus nidulans Copper Amine Oxidase. %B Biochemistry %D 2011 %C United States %I American Chemical Society %V 50 %N 25 %P 5718-30 %@ 0006-2960 %X Aspergillus nidulans amine oxidase (ANAO) has the unusual ability among the family of copper and trihydroxyphenylalanine quinone-containing amine oxidases of being able to oxidize the amine side chains of lysine residues in large peptides and proteins. We show here that in common with the related enzyme from the yeast Pichia pastoris, ANAO can promote the cross-linking of tropoelastin and oxidize the lysine residues in ??-casein proteins and tropoelastin. The crystal structure of ANAO, the first for a fungal enzyme in this family, has been determined to a resolution of 2.4 ??. The enzyme is a dimer with the archetypal fold of a copper-containing amine oxidase. The active site is the most open of any of those of the structurally characterized enzymes in the family and provides a ready explanation for its lysine oxidase-like activity. %Z FOR Codes: 60112 %0 Journal Article %~ PubMed %A Rnjak-Kovacina, Jelena %A Wise, Steven G %A Li, Zhe %A Maitz, Peter K M %A Young, Cara J %A Wang, Yiwei %A Weiss, Anthony S %T Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering. %B Biomaterials %D 2011 %C Netherlands %I Elsevier BV %V 32 %N 28 %P 6729-36 %@ 0142-9612 %X We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young''s moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis. %Z FOR Codes: 60106 100404 %0 Journal Article %~ PubMed %A Annabi, Nasim %A Fathi, Ali %A Mithieux, Suzanne M %A Martens, Penny %A Weiss, Anthony S %A Dehghani, Fariba %T The effect of elastin on chondrocyte adhesion and proliferation on poly (ɛ-caprolactone)/elastin composites. %B Biomaterials %D 2011 %C Netherlands %I Elsevier BV %V 32 %N 6 %P 1517-25 %@ 0142-9612 %X The aim of this study was to demonstrate the effect of elastin on chondrocyte adhesion and proliferation within the structure of poly (??-caprolactone) (PCL)/elastin composites. The homogenous 3D structure composites were constructed by using high pressure CO(2) in two stages. Porous PCL structures with average pore sizes of 540 ?? 21 ??m and a high degree of interconnectivity were produced using gas foaming/salt leaching. The PCL scaffolds were then impregnated with elastin and cross-linked with glutaraldehyde (GA) under high pressure CO(2). The effects of elastin and cross-linker concentrations on the characteristics of composites were investigated. Increasing the elastin concentration from 25mg/ml to 100mg/ml elevated the amount of cross-linked elastin inside the macropores of PCL. Fourier transform infrared (FTIR) analysis showed that elastin was homogeneously distributed throughout the 3D structure of all composites. The weight gain of composites increased 2-fold from 15.8 ?? 0.3 to 38.3 ?? 0.7 (w/w) % by increasing the elastin concentration from 25mg/ml to 50mg/ml and approached a plateau above this concentration. The presence of elastin within the pores of PCL improved the water uptake properties of PCL scaffolds; the water uptake ratio of PCL was enhanced 100-fold from 0.030 ?? 0.005g liquid/g polymer to 11.80 ?? 0.01g liquid/g polymer, when the elastin solution concentration was 50mg/ml. These composites exhibited lower compressive modulus and energy loss compared to pure PCL scaffolds due to their higher water content and elasticity. In vitro studies show that these composites can support primary articular cartilage chondrocyte adhesion and proliferation within the 3D structures. These results demonstrate the potential of using PCL/elastin composites for cartilage repair. %Z FOR Codes: 90301 %0 Journal Article %~ PubMed %A Hu, Xiao %A Park, Sang-Hyug %A Gil, Eun Seok %A Xia, Xiao-Xia %A Weiss, Anthony S %A Kaplan, David L %T The influence of elasticity and surface roughness on myogenic and osteogenic-differentiation of cells on silk-elastin biomaterials. %B Biomaterials %D 2011 %C Netherlands %I Elsevier BV %V 32 %N 34 %P 8979-8989 %@ 0142-9612 %X 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. %Z FOR Codes: 60106 %0 Journal Article %A Bax, Daniel V. %A Liu, Siyuan John %A McKenzie, David R. %A Bilek, Marcela M. M. %A Weiss, Anthony %T Tropoelastin Switch and Modulated Endothelial Cell Binding to PTFE %B BioNanoScience %D 2011 %C United States %I Springer New York LLC %V 1 %N %P 123-127 %@ 2191-1630 %X %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Hu, Xiao %A Wang, Xiuli %A Rnjak, Jelena %A Weiss, Anthony S %A Kaplan, David L %T Biomaterials derived from silk-tropoelastin protein systems. %B Biomaterials %D 2010 %C Netherlands %I Elsevier BV %V 31 %N 32 %P 8121-8131 %@ 0142-9612 %X A structural protein blend system based on silkworm silk fibroin and recombinant human tropoelastin is described. Silk fibroin, a semicrystalline fibrous protein with beta-sheet crystals provides mechanical strength and controllable biodegradation, while tropoelastin, a noncrystallizable elastic protein provides elasticity. Differential scanning calorimetry (DSC) and temperature modulated DSC (TMDSC) indicated that silk becomes miscible with tropoelastin at different blend ratios, without macrophase separation. Fourier transform infrared spectroscopy (FTIR) revealed secondary structural changes of the blend system (beta-sheet content) before and after methanol treatment. Atomic Force Microscopy (AFM) nano-indentation demonstrated that blending silk and tropoelastin at different ratios resulted in modification of mechanical features, with resilience from approximately 68%- approximately 97%, and elastic modulus between 2 and 9 Mpa, depending on the ratio of the two polymers. Some of these values are close to those of native aortic elastin or elastin-like polypeptides. Significantly, during blending and drying silk-tropoelastin form micro- and nano-scale porous morphologies which promote human mesenchymal stem cell attachment and proliferation. These blends offer a new protein biomaterial system for cell support and tailored biomaterial properties to match mechanical needs. %Z FOR Codes: 30406 90301 %0 Journal Article %~ PubMed %A Annabi, Nasim %A Mithieux, Suzanne M %A Weiss, Anthony S %A Dehghani, Fariba %T Cross-linked open-pore elastic hydrogels based on tropoelastin, elastin and high pressure CO(2). %B Biomaterials %D 2010 %C Netherlands %I Elsevier BV %V 31 %N 7 %P 1655-65 %@ 0142-9612 %X In this study the effect of high pressure CO(2) on the synthesis and characteristics of elastin-based hybrid hydrogels was investigated. Tropoelastin/alpha-elastin hybrid hydrogels were fabricated by chemically cross-linking tropoelastin/alpha-elastin solutions with glutaraldehyde at high pressure CO(2). Dense gas CO(2) had a significant impact on the characteristics of the fabricated hydrogels including porosity, swelling ratio, compressive properties, and modulus of elasticity. Compared to fabrication at atmospheric pressure high pressure CO(2) based construction eliminated the skin-like formation on the top surfaces of hydrogels and generated larger pores with an average pore size of 78 +/- 17 microm. The swelling ratios of composite hydrogels fabricated at high pressure CO(2) were lower than the gels produced at atmospheric pressure as a result of a higher degree of cross-linking. Dense gas CO(2) substantially increased the mechanical properties of fabricated hydrogels. The compressive and tensile modulus of 50/50 weight ratio tropoelastin/alpha-elastin composite hydrogels were enhanced 2 and 2.5 fold, respectively, when the pressure was increased from 1 to 60 bar. In vitro studies show that the presence of large pores throughout the hydrogel matrix fabricated at high pressure CO(2) enabled the migration of human skin fibroblast cells 300 microm into the construct. %Z FOR Codes: 100404 %0 Journal Article %~ Isi %A Heinz, A. %A Jung, M. C. %A Duca, L. %A Sippl, W. %A Taddese, S. %A Ihling, C. %A Rusciani, A. %A Jahreis, G. %A Weiss, A. S. %A Neubert, R. H. H. %A Schmelzer, C. E. H. %T Degradation of tropoelastin by matrix metalloproteinases - cleavage site specificities and release of matrikines %B FEBS Journal %D 2010 %C United Kingdom, Denm %I Wiley-Blackwell Publishing Ltd. %V 277 %N 8 %P 1939-1956 %@ 1742-464X %X %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Almine, Jessica F %A Bax, Daniel V %A Mithieux, Suzanne M %A Nivison-Smith, Lisa %A Rnjak, Jelena %A Waterhouse, Anna %A Wise, Steven G %A Weiss, Anthony S %T Elastin-based materials. %B Chemical Society reviews %D 2010 %C United Kingdom %I Royal Society of Chemistry %V 39 %N 9 %P 3371-9 %@ 1460-4744 %X Elastin is a versatile elastic protein that dominates flexible tissues capable of recoil, and facilitates commensurate cell interactions in these tissues in all higher vertebrates. Elastin''s persistence and insolubility hampered early efforts to construct versatile biomaterials. Subsequently the field has progressed substantially through the adapted use of solubilized elastin, elastin-based peptides and the increasing availability of recombinant forms of the natural soluble elastin precursor, tropoelastin. These interactions allow for the formation of a sophisticated range of biomaterial constructs and composites that benefit from elastin''s physical properties of innate assembly and elasticity, and cell interactive properties as discussed in this tutorial review. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Dyksterhuis, L B %A Weiss, A S %T Homology models for domains 21-23 of human tropoelastin shed light on lysine crosslinking. %B Biochemical and biophysical research communications %D 2010 %C United States %I Academic Press %V 396 %N 4 %P 870-873 %@ 0006-291X %X The contiguous crosslinking domain at the center of human tropoelastin encoded by exons 21-23 contains an unusual ''hinge'' region thought to adopt both open and closed conformations. Key lysines 425 and 437 have been implicated in both artificial and lysyl oxidase mediated crosslinks. We have examined the importance of hinge conformation to the proximity of these lysines and their ability to undergo intramolecular and intermolecular crosslinks using homology models. The results, counter intuitively, indicate that the more open hinge conformations favor intramolecular crosslinking, while the more closed conformations favor intermolecular crosslinking. We also present evidence that the sidechains of lysines 425 and 437 are able to make direct contact enabling an intramolecular lysyl oxidase mediated crosslink. %Z FOR Codes: 30406 %0 Journal Article %~ PubMed %A Waterhouse, Anna %A Bax, Daniel V %A Wise, Steven G %A Yin, Yongbai %A Dunn, Louise L %A Yeo, Giselle C %A Ng, Martin K C %A Bilek, Marcela M M %A Weiss, Anthony S %T Stability of a Therapeutic Layer of Immobilized Recombinant Human Tropoelastin on a Plasma-Activated Coated Surface. %B Pharmaceutical Research %D 2010 %C United States %I Springer New York LLC %V 28 %N 6 %P 1415-1421 %@ 1573-904X %X 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. %Z FOR Codes: 90301 %0 Journal Article %~ PubMed %A Tu, Yidong %A Wise, Steven G %A Weiss, Anthony S %T Stages in tropoelastin coalescence during synthetic elastin hydrogel formation. %B Micron (Oxford, England : 1993) %D 2010 %C United Kingdom %I Pergamon %V 41 %N 3 %P 268-72 %@ 1878-4291 %X Synthetic human tropoelastin was chemically cross-linked to form elastic hydrogel-like structures in vitro. Discrete stages were identified during elastic hydrogel formation by cross-linking tropoelastin with bis(sulfosuccinimidyl) suberate at a range of protein concentrations during this process. In the early stages of this process, particles with the same dimensions as tropoelastin were seen. As hydrogel formation progressed, monomer width fibres were also observed. Overall, four distinct stages were identified: (1) tropoelastin monomers form discrete particles in the order of 200 nm diameter, (2) these particles merge to form larger spheres, (3) spheres coalesce into open linked networks, (4) coalesced spheres consolidate to form a porous structure to give synthetic elastin hydrogels. %Z FOR Codes: 60112 %0 Journal Article %~ PubMed %A Muiznieks, Lisa D %A Weiss, Anthony S %A Keeley, Fred W %T Structural disorder and dynamics of elastin. %B Biochemistry and cell biology = Biochimie et biologie cellulaire %D 2010 %C Canada %I NRC Research Press %V 88 %N 2 %P 239-250 %@ 1208-6002 %X Elastin is a self-assembling, extracellular-matrix protein that is the major provider of tissue elasticity. Here we review structural studies of elastin from over four decades, and draw together evidence for solution flexibility and conformational disorder that is inherent in all levels of structural organization. The characterization of disorder is consistent with an entropy-driven mechanism of elastic recoil. We conclude that conformational disorder is a constitutive feature of elastin structure and function. %Z FOR Codes: 30406 %0 Journal Article %~ PubMed %A Holst, Jeff %A Watson, Sarah %A Lord, Megan S %A Eamegdool, Steven S %A Bax, Daniel V %A Nivison-Smith, Lisa B %A Kondyurin, Alexey %A Ma, Liang %A Oberhauser, Andres F %A Weiss, Anthony S %A Rasko, John E J %T Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells. %B Nature biotechnology %D 2010 %C United States %I Nature Publishing Group %V 28 %N 10 %P 1123-8 %@ 1546-1696 %X Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture. %Z FOR Codes: 60103 100404 %0 Journal Article %~ PubMed %A Tu, Yidong %A Mithieux, Suzanne M %A Annabi, Nasim %A Boughton, Elizabeth A %A Weiss, Anthony S %T Synthetic elastin hydrogels that are coblended with heparin display substantial swelling, increased porosity, and improved cell penetration. %B Journal of biomedical materials research. Part A %D 2010 %C United States %I John Wiley & Sons, Inc. %V 95 %N 4 %P 1215-22 %@ 1552-4965 %X Synthetic elastin hydrogels are useful tissue engineering scaffolds because they present cell binding sequences and display physical performance similar to that of human elastic tissue. Small pores and a low porosity can limit cellular penetration into elastin scaffolds. To overcome this problem, glycosaminoglycans were coblended with tropoelastin during the formation of synthetic elastin hydrogels. Heparin and dermatan sulfate increased the pore size and porosity of the hydrogels. Heparin was particularly effective as it enlarged the pore size from 6.6 ?? 2.1 ??m to 23.8 ?? 8.5 ??m, and generated structures occasionally separated by finely fenestrated thin walls, which allowed human dermal fibroblast cells to migrate as deep as ???300 ??m into the hydrogel under diffusion-limiting static culture conditions. Most cells displayed spindle-like morphology, appeared histologically normal and presented intact nuclei, as expected for a viable population. Hydrogel swelling studies showed that each of the hydrogels contracted as the temperature was raised from 4??C to 37??C; synthetic elastin-heparin was least affected by temperature with a contraction of only 22.4 ?? 1.2%, which would facilitate its transition from cold storage to body temperature. All hydrogels displayed similar compression moduli of 5.5 ?? 0.4 to 6.9 ?? 0.6 kPa. Compressive elastic energy losses for synthetic elastin-heparin and synthetic elastin were 33.7 ?? 1.3% and 31.7 ?? 2.2% respectively. %Z FOR Codes: 90301 %0 Journal Article %~ PubMed %A Waterhouse, Anna %A Yin, Yongbai %A Wise, Steven G %A Bax, Daniel V %A McKenzie, David R %A Bilek, Marcela M M %A Weiss, Anthony S %A Ng, Martin K C %T The immobilization of recombinant human tropoelastin on metals using a plasma-activated coating to improve the biocompatibility of coronary stents. %B Biomaterials %D 2010 %C Netherlands %I Elsevier BV %V 31 %N 32 %P 8332-40 %@ 0142-9612 %X Current endovascular stents have sub-optimal biocompatibility reducing their clinical efficacy. We previously demonstrated a plasma-activated coating (PAC) that covalently bound recombinant human tropoelastin (TE), a major regulator of vascular cells in vivo, to enhance endothelial cell interactions. We sought to develop this coating to enhance its mechanical properties and hemocompatibility for application onto coronary stents. The plasma vapor composition was altered by incorporating argon, nitrogen, hydrogen or oxygen to modulate coating properties. Coatings were characterized for 1) surface properties, 2) mechanical durability, 3) covalent protein binding, 4) endothelial cell interactions and 5) thrombogenicity. The N(2)/Ar PAC had optimal mechanical properties and did not delaminate after stent expansion. The N(2)/Ar PAC was mildly hydrophilic and covalently bound the highest proportion of TE, which enhanced endothelial cell proliferation. Acute thrombogenicity was assessed in a modified Chandler loop using human blood. Strikingly, the N(2)/Ar PAC alone reduced thrombus weight by ten-fold compared to 316L SS, a finding unaltered with immobilized TE. Serum soluble P-selectin was reduced on N(2)/Ar PAC and N(2)/Ar PAC + TE (p < 0.05), consistent with reduced platelet activation. We have demonstrated a coating for metal alloys with multifaceted biocompatibility that resists delamination and is non-thrombogenic, with implications for improving coronary stent efficacy. %Z FOR Codes: 100403 %0 Journal Article %~ PubMed %A Naumann, Christoph %A Mithieux, Suzanne M %A Szekely, David %A Tu, Yidong %A Weiss, Anthony S %A Kuchel, Philip W %T "Setting paint" analogy for the hydrophobic self-association of tropoelastin into elastin-like hydrogel. %B Biopolymers %D 2009 %C United States %I John Wiley & Sons, Inc. %V 91 %N 5 %P 321-330 %@ 0006-3525 %X Alkaline tropoelastin solutions (pH 11) were optically clear at low temperatures, but a firm gel formed when the temperature was raised to 37 degrees C. Reversion to a clear solution took place if the temperature was lowered to below 20 degrees C within less than 2 h, but not if 37 degrees C was maintained for several hours. The precipitated elastin-like hydrogel thus formed did not visually redissolve at low temperatures. Tropoelastin hydrogel was stable to subsequent washings with alkaline solution at 37 degrees C, but at 4 degrees C some hydrogel redissolved showing that association is at least partly reversible. Washing the hydrogel with neutral 8M urea solution at 4 degrees C dissolved less than 10% of tropoelastin in 24 h. We characterized this phenomenon by combining temperature-controlled light microscopy analysis, 1H NMR spectroscopy (temperature, diffusion, and relaxation time studies), and UV-absorption-based concentration measurements. The self-association of tropoelastin at pH 11 is due to hydrophobic interactions in an emulsion-like system in which the spherules coalesce in a manner like a water-based latex paint that forms a durable hydrophobic sheet as water and the organic solvent evaporate. In the present case, the sedimentation and entanglement of the tropoelastin porous sheets means that reverse dissolution is a kinetically slow process. %Z FOR Codes: 60112 %0 Journal Article %~ PubMed %A Bax, Daniel V %A Rodgers, Ursula R %A Bilek, Marcela M M %A Weiss, Anthony S %T Cell adhesion to tropoelastin is mediated via the C-terminal GRKRK motif and integrin alphaVbeta3. %B The Journal of biological chemistry %D 2009 %C United States %I American Society for Biochemistry and Molecular Bi %V 284 %N 42 %P 28616-23 %@ 1083-351X %X Elastin fibers are predominantly composed of the secreted monomer tropoelastin. This protein assembly confers elasticity to all vertebrate elastic tissues including arteries, lung, skin, vocal folds, and elastic cartilage. In this study we examined the mechanism of cell interactions with recombinant human tropoelastin. Cell adhesion to human tropoelastin was divalent cation-dependent, and the inhibitory anti-integrin alpha(V)beta(3) antibody LM609 inhibited cell spreading on tropoelastin, identifying integrin alpha(V)beta(3) as the major fibroblast cell surface receptor for human tropoelastin. Cell adhesion was unaffected by lactose and heparin sulfate, indicating that the elastin-binding protein and cell surface glycosaminoglycans are not involved. The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cation-dependent manner, identifying this as an integrin binding motif required for cell adhesion. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Yin, Yongbai %A Wise, Steven G %A Nosworthy, Neil J %A Waterhouse, Anna %A Bax, Daniel V %A Youssef, Hani %A Byrom, Michael J %A Bilek, Marcela M M %A McKenzie, David R %A Weiss, Anthony S %A Ng, Martin K C %T Covalent immobilisation of tropoelastin on a plasma deposited interface for enhancement of endothelialisation on metal surfaces. %B Biomaterials %D 2009 %C United Kingdom %I Elsevier Sci Ltd %V 30 %N 9 %P 1675-1681 %@ 0142-9612 %X Currently available endovascular metallic implants such as stents exhibit suboptimal biocompatibility in that they re-endothelialise poorly leaving them susceptible to thrombosis. To improve the interaction of these implants with endothelial cells we developed a surface coating technology, enabling the covalent attachment of biomolecules to previously inert metal surfaces. Using horseradish peroxidase as a probe, we demonstrate that the polymerised surface can retain the presentation and activity of an immobilised protein. We further demonstrated the attachment of tropoelastin, an extracellular matrix protein critical to the correct arrangement and function of vasculature. Not only it is structurally important, but it plays a major role in supporting endothelial cell growth, while modulating smooth muscle cell infiltration. Tropoelastin was shown to bind to the surface in a covalent monolayer, supplemented with additional physisorbed multilayers on extended incubation. The physisorbed tropoelastin layers can be washed away in buffer or SDS while the first layer of tropoelastin remains tightly bound. The plasma coated stainless steel surface with immobilised tropoelastin was subsequently found to have improved biocompatibility by promoting endothelial cell attachment and proliferation relative to uncoated stainless steel controls. Tropoelastin coatings applied to otherwise inert substrates using this technology could thus have broad applications to a range of non-polymeric vascular devices. %Z FOR Codes: 90301 90304 %0 Journal Article %~ Isi %A Yin, Y. B. %A Fisher, K. %A Nosworthy, N. J. %A Bax, D. %A Rubanov, S. %A Gong, B. %A Weiss, A. S. %A McKenzie, D. R. %A Bilek, M. M. M. %T Covalently Bound Biomimetic Layers on Plasma Polymers with Graded Metallic Interfaces for in vivo Implants %B Plasma Processes and Polymers %D 2009 %C Germany %I WILEY-V C H VERLAG %V 6 %N 10 %P 658-666 %@ 1612-8850 %X %Z FOR Codes: 1004 %0 Journal Article %A Wise, Steven %A Mithieux, Suzanne %A Weiss, Anthony %T Engineered tropoelastin and elastin-based biomaterials %B Advances in Protein Chemistry and Structural Biology %D 2009 %C United States %I Academic Press %V 78 %N %P 1-24 %@ 1876-1623 %X %Z FOR Codes: 30406 %0 Journal Article %~ PubMed %A Mithieux, Suzanne M %A Tu, Yidong %A Korkmaz, Emine %A Braet, Filip %A Weiss, Anthony S %T In situ polymerization of tropoelastin in the absence of chemical cross-linking. %B Biomaterials %D 2009 %C UK %I Pergamon %V 30 %N 0 %P 431-5 %@ 0142-9612 %X Tropoelastin, the polypeptide monomer precursor of elastin, is covalently cross-linked to give stable elastic structures. We show here that elastic biomaterials can be generated from tropoelastin in the absence of the classically accepted cross-linking pathway. Under alkaline conditions tropoelastin proceeds through a sol-gel transition leading to the formation of an irreversible hydrogel. This does not occur at neutral pH. The resulting biomaterial is stable, elastic and flexible. Scanning electron microscopy revealed that the hydrogel forms through the coalescence of approximately 1 microm quantized protein spheres. These spheres resemble the tropoelastin-rich globules that accumulate on cultured cell surfaces during elastin formation. In vitro cell culture studies demonstrate that the hydrogel can support human skin fibroblast proliferation. In vivo studies demonstrate that following injection, the tropoelastin solution undergoes rapid localized gelation to form a persistent mass. These subcutaneous rodent injection data establish the material''s potential as a novel cell-compatible elastic scaffold that can be formed in situ. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Taddese, Samuel %A Weiss, Anthony S %A Jahreis, Günther %A Neubert, Reinhard H H %A Schmelzer, Christian E H %T In vitro degradation of human tropoelastin by MMP-12 and the generation of matrikines from domain 24. %B Matrix biology : journal of the International Society for Matrix Biology %D 2009 %C Netherlands %I Elsevier BV %V 28 %N 2 %P 84-91 %@ 1569-1802 %X Degradation of elastic fibers in tissues can result in the development of disorders that include aneurysms, atherosclerosis, and loss of skin elasticity. Tropoelastin is the precursor of the cross-linked elastin and its expression is triggered by elastin-degrading factors as a response to damage. Factors like UV radiation not only increase the expression of tropoelastin but also potent metalloelastases such as macrophage elastase (MMP-12). The development of elastin-degrading diseases, moreover, is a chronic process during which elastin and tropoelastin are repeatedly exposed to attacks by MMP-12. Hence, in this work we report the in vitro susceptibility of tropoelastin and the potential of MMP-12 to generate matrikines. This work provides evidence that tropoelastin is substantially and rapidly degraded by MMP-12 even at very dilute enzyme concentrations. MMP-12 cleaves at least 86 sites in tropoelastin. Analysis of the generated peptides revealed that some small peptides contained the motif GXXPG that may enable them to bind with the elastin binding protein (EBP). Furthermore, using synthesized peptides it was confirmed that several sites in the sequence encoded by exon 24 which contains repetitive units of biologically active VGVAPG domains are susceptible to attack by MMP-12, provided that the active subsites in MMP-12 (S(4) to S(4)'') are occupied. Such cleavage events have lead to the generation of ligands that may bind to EBP. %Z FOR Codes: 60103 %0 Journal Article %~ PubMed %A Yang, Shaoguang %A Xie, Yuxuan %A Yang, Wenrong %A Zheng, Rongkun %A Stevens, Frankie %A Korkmaz, Emine %A Weiss, Anthony S %A Ringer, Simon P %A Braet, Filip %T In vitro studies of cells grown on the superconductor PrO(x)FeAs. %B Micron (Oxford, England : 1993) %D 2009 %C United Kingdom %I Pergamon %V 40 %N 4 %P 476-479 %@ 1878-4291 %X The recent discovery of arsenic-based high temperature superconductors has reignited interest in the study of superconductor: biological interfaces. However, the new superconductor materials involve the chemistry of arsenic and their toxicity remains unclear [Hand, E., 2008. Nature 452 (24), 922]. In this study the possible adverse effects of this new family of superconductors on cells have been examined. Cell culture studies in conjunction with microscopy and viability assays were employed to examine the influence of arsenic-based superconductor PrO(x)FeAs (x=0.75) material in vitro. Imaging data revealed that cells were well adhered and spread on the surface of the superconductor. Furthermore, cytotoxicity studies showed that cells were unaffected during the time-course of the experiments, providing support for the biocompatibility aspects of PrO(x)FeAs-based superconductor material. %Z FOR Codes: 60103 %0 Journal Article %~ PubMed %A Bax, Daniel V %A McKenzie, David R %A Weiss, Anthony S %A Bilek, Marcela M M %T Linker-free covalent attachment of the extracellular matrix protein tropoelastin to a polymer surface for directed cell spreading. %B Acta biomaterialia %D 2009 %C Netherlands %I Elsevier BV %V 5 %N 0 %P 3371-81 %@ 1878-7568 %X Polymers are used for the fabrication of many prosthetic implants. It is desirable for these polymers to promote biological function by promoting the adhesion, differentiation and viability of cells. Here we have used plasma immersion ion implantation (PIII) treatment of polystyrene to modify the polymer surface, and so modulate the binding of the extracellular matrix protein tropoelastin. PIII treated, but not untreated polystyrene, bound tropoelastin in a sodium dodecyl sulfate (SDS)-resistant manner, consistent with previous enzyme-binding data that demonstrated the capability of these surfaces to covalently attach proteins without employing chemical linking molecules. Furthermore sulfo-NHS acetate (SNA) blocking of tropoelastin lysine side chains eliminated the SDS-resistant binding of tropoelastin to PIII-treated polystyrene. This implies tropoelastin is covalently attached to the PIII-treated surface via its lysine side chains. Cell spreading was only observed on tropoelastin coated, PIII-treated polystyrene surfaces, indicating that tropoelastin was more biologically active on the PIII-treated surface compared to the untreated surface. A contact mask was used to pattern the PIII treatment. Following tropoelastin attachment, cells spread preferentially on the PIII-treated sections of the polystyrene surface. This demonstrates that PIII treatment of polystyrene improves the polymer''s tropoelastin binding properties, with advantages for tissue engineering and prosthetic design. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Rnjak, Jelena %A Li, Zhe %A Maitz, Peter K M %A Wise, Steven G %A Weiss, Anthony S %T Primary human dermal fibroblast interactions with open weave three-dimensional scaffolds prepared from synthetic human elastin. %B Biomaterials %D 2009 %C Netherlands %I Elsevier BV %V 30 %N 32 %P 6469-77 %@ 0142-9612 %X We present an elastic, fibrous human protein-based and cell-interactive dermal substitute scaffold based on synthetic human elastin. Recombinant human tropoelastin promoted primary human dermal fibroblast attachment, spreading and proliferation. Tropoelastin was cross-linked to form a synthetic elastin (SE) hydrogel matrix and electrospun into fibrous SE scaffolds. Fibroblasts attached to and proliferated across SE hydrogel scaffold surfaces for at least 14 days and deposited the extracellular matrix proteins fibronectin and collagen type I. To allow for the benefit of greater cell infiltration, SE was electrospun into open weave, fibrous scaffolds that closely mimic the fibrous nature of the skin dermis. 3D SE scaffolds were robust and consisted of flat, ribbon-like fibers with widths that are similar to native dermal elastic fibers. The scaffolds displayed elasticity close to that of natural elastin. 3D SE retained the ability to interact with primary human dermal fibroblasts, which consistently attached and proliferated to form monolayers spanning the entire scaffold surface. The open weave design, with larger spaces between individual fibers and greater fiber diameters beneficially allowed for substantial cell infiltration throughout the scaffolds. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Annabi, Nasim %A Mithieux, Suzanne M %A Boughton, Elizabeth A %A Ruys, Andrew J %A Weiss, Anthony S %A Dehghani, Fariba %T Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro. %B Biomaterials %D 2009 %C Netherlands %I Elsevier BV %V 30 %N 27 %P 4550-7 %@ 0142-9612 %X In this study the feasibility of using high pressure CO2 to produce porous alpha-elastin hydrogels was investigated. Alpha-elastin was chemically crosslinked with hexamethylene diisocyanate that can react with various functional groups in elastin such as lysine, cysteine, and histidine. High pressure CO2 substantially affected the characteristics of the fabricated hydrogels. The pore size of the hydrogels was enhanced 20-fold when the pressure was increased from 1 bar to 60 bar. The swelling ratio of the samples fabricated by high pressure CO2 was also higher than the gels produced under atmospheric pressure. The compression modulus of alpha-elastin hydrogels was increased as the applied strain magnitude was modified from 40% to 80%. The compression modulus of hydrogels produced under high pressure CO2 was 3-fold lower than the gels formed at atmospheric conditions due to the increased porosity of the gels produced by high pressure CO2. The fabrication of large pores within the 3D structures of these hydrogels substantially promoted cellular penetration and growth throughout the matrices. The highly porous alpha-elastin hydrogel structures fabricated in this study have potential for applications in tissue engineering. %Z FOR Codes: 90301 %0 Journal Article %~ PubMed %A Nivison-Smith, Lisa %A Rnjak, Jelena %A Weiss, Anthony S %T Synthetic human elastin microfibers: Stable cross-linked tropoelastin and cell interactive constructs for tissue engineering applications. %B Acta biomaterialia %D 2009 %C Netherlands %I Elsevier BV %V 6 %N 0 %P 354-9 %@ 1878-7568 %X Elastin is a key extracellular matrix protein in a range of tissues and a viable candidate for elastic tissue engineering. Elastin is not typically incorporated into engineered scaffolds because of lack of access to pure, homogeneous human elastin. Recombinant human tropoelastin, the monomer precursor of elastin, can be chemically cross-linked to form a polymer and used to generate biomaterials with physical properties similar to native elastin. In this study, we use electrospinning to generate versatile tropoelastin microfibers. Tropoelastin retained structural and biological properties, including secondary structure and coacervation temperature, after fiber formation but was solubilized on exposure to an aqueous environment prior to cross-linking. Two cross-linking methods were utilized to generate synthetic elastin microfibers that were stable in aqueous environments. Microfibers stably persisted for up to 180 days at 37 degrees C. Three primary human cell types derived from elastic tissues were assessed and found to attach and proliferate on both types of microfibers. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Annabi, Nasim %A Mithieux, Suzanne M %A Weiss, Anthony S %A Dehghani, Fariba %T The fabrication of elastin-based hydrogels using high pressure CO(2). %B Biomaterials %D 2009 %C UK %I Pergamon %V 30 %N 0 %P 1-7 %@ 0142-9612 %X The aim of this study was to investigate the effect of high pressure CO(2) on the crosslinking of elastin-based polymers and the characteristics of the fabricated hydrogels. A hydrogel was fabricated by chemically crosslinking alpha-elastin with glutaraldehyde at high pressure CO(2). The effects of pressure, reaction time, and crosslinker concentration on the characteristics of the fabricated hydrogels were determined. The reaction time had negligible effect on either the swelling ratio or the pore size of the fabricated hydrogels. Increasing the processing pressure from 30bar to 150bar resulted in a 60% increase in the hydrogel swelling ratio. The crosslinked hydrogels displayed stimuli-responsive characteristics towards temperature and salt concentration. The dense gas process facilitated coacervation, expedited the crosslinking reaction, and dramatically affected the micro- and macrostructures of pores within the sample. The results of micro-CT scan and SEM images demonstrated that pore interconnectivity was substantially enhanced for alpha-elastin hydrogels fabricated using high pressure CO(2). Dense gas CO(2) reduced the wall thickness and size of the pores and importantly induced channels within the structure of the alpha-elastin hydrogels. In vitro cell culture studies demonstrated that the channels facilitated fibroblast penetration and proliferation within alpha-elastin structures. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Bax, Daniel V %A McKenzie, David R %A Weiss, Anthony S %A Bilek, Marcela M M %T The linker-free covalent attachment of collagen to plasma immersion ion implantation treated polytetrafluoroethylene and subsequent cell-binding activity. %B Biomaterials %D 2009 %C Netherlands %I Elsevier BV %V 31 %N 9 %P 2526-34 %@ 0142-9612 %X It is desirable that polymers used for the fabrication of prosthetic implants promote biological functions such as cellular adhesion, differentiation and viability. In this study, we have used plasma immersion ion implantation (PIII) to modify the surface of polytetrafluoroethylene (PTFE), thereby modulating the binding mechanism of collagen. The amount of collagen bound to the polymer surface following PIII-treatment was similar to that bound by non-covalent physisorption. In a manner consistent with previous enzyme and tropoelastin binding data, the collagen bound to the PIII-treated PTFE surface was resistant to sodium dodecyl sulfate (SDS) elution whilst collagen bound to the untreated surface was fully removed. This demonstrates the capability of PIII-treated surfaces to covalently attach collagen without employing chemical linking molecules. Only the collagen bound to the PIII-treated PTFE surface supported human dermal fibroblast attachment and spreading. This indicates that collagen on the PIII-treated surface possesses increased adhesive activity as compared to that on the untreated surface. Cell adhesion was inhibited by EDTA when the collagen was bound to PIII-treated PTFE, as expected for integrin involvement. Additionally this adhesion was sensitive to the conformation of the bound collagen. Increased actin cytoskeletal assembly was observed on cells spreading onto collagen-coated PIII-treated PTFE compared to the collagen-coated untreated PTFE. These data demonstrate the retention of collagen''s biological properties following its attachment to PIII-treated PTFE, suggesting advantages for tissue engineering and prosthetic design. %Z FOR Codes: 100404 %0 Journal Article %~ PubMed %A Tu, Yidong %A Weiss, Anthony S %T Transient tropoelastin nanoparticles are early-stage intermediates in the coacervation of human tropoelastin whose aggregation is facilitated by heparan sulfate and heparin decasaccharides. %B Matrix biology : journal of the International Society for Matrix Biology %D 2009 %C Netherlands %I Elsevier BV %V 29 %N 0 %P 152-9 %@ 1569-1802 %X Tropoelastin assembly is a key step in the formation of elastin. We consider how nanoscale intracellular assemblies of tropoelastin can congregate in an extracellular environment to give microscale aggregates. We describe novel 200-300 nm spherical particles that serve as intermediates in the formation of the coacervate. Their aggregation gives 800 nm to 1 microm species. This process is facilitated by heparan sulfate and dermatan sulfate interactions which effectively lower the critical concentration to facilitate this transition. This coacervation process was examined using a panel of heparin chains of various lengths and showed greatest efficacy for the decasaccharide, followed by the octasaccharide, while the hexasaccharide displayed the shortest efficacious length. We propose that these oligosaccharide interactions enable the charge-mediated aggregation of positively charged tropoelastin. This biochemistry models glycosaminoglycan interactions on the cell surface during elastogenesis which is characterized by the clustering of nascent tropoelastin aggregates to form micron-sized spherules. %Z FOR Codes: 60106 %0 Journal Article %~ PubMed %A Dyksterhuis, Leanne B %A Carter, Elizabeth A %A Mithieux, Suzanne M %A Weiss, Anthony S %T Tropoelastin as a thermodynamically unfolded premolten globule protein: The effect of trimethylamine N-oxide on structure and coacervation. %B Archives of biochemistry and biophysics %D 2009 %C United States %I Academic Press %V 487 %N 2 %P 79-84 %@ 1096-0384 %X Tropoelastin is the monomer building block of the biopolymer elastin, which is responsible for elasticity in arteries, lung and skin. Previous studies have shown that, in contrast to predictions made based on primary sequence, tropoelastin has little regular secondary structure in aqueous solution and displays considerable flexibility. This investigation defines the level of residual structure present in tropoelastin and uses the naturally-occurring structure-inducing osmolyte trimethylamine N-oxide to examine the potential for regular structure in tropoelastin. Tropoelastin is defined as a thermodynamically unfolded premolten globule, which can account for its ability to elastically deform. %Z FOR Codes: 110199 %0 Journal Article %~ PubMed %A Wise, Steven G %A Weiss, Anthony S %T Tropoelastin. %B The international journal of biochemistry & cell biology %D 2009 %C United Kingdom %I Pergamon %V 41 %N 0 %P 494-7 %@ 1357-2725 %X Tropoelastin is a 60-72 kDa alternatively spliced extracellular matrix protein and a key component of elastic fibres. It is found in all vertebrates except for cyclostomes. Secreted tropoelastin is tethered to the cell surface, where it aggregates into organised spheres for cross-linking and incorporation into growing elastic fibres. Tropoelastin is characterised by alternating hydrophobic and hydrophilic domains and is highly flexible. The conserved C-terminus is an area of the molecule of particular biological importance in that it is required for both incorporation into elastin and for cellular interactions. Mature cross-linked tropoelastin gives elastin, which confers resilience and elasticity on a diverse range of tissues. Elastin gene disruptions in disease states and knockout mice emphasise the importance of proper tropoelastin production and assembly, particularly in vascular tissue. Tropoelastin constructs hold promise as biomaterials as they mimic many of elastin''s physical and biological properties with the capacity to replace damaged elastin-rich tissue. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Macdonald, Christopher %A Morrow, Richard %A Weiss, Anthony S %A Bilek, Marcela M M %T Covalent attachment of functional protein to polymer surfaces: a novel one-step dry process. %B Journal of the Royal Society, Interface / the Royal Society %D 2008 %C United Kingdom %I The Royal Society %V 5 %N %P 663-9 %@ 1742-5689 %X The attachment of bioactive protein to surfaces underpins the development of biosensors and diagnostic microarrays. We present a surface treatment using plasma immersion ion implantation (PIII) to create stable covalent binding sites for the attachment of functional soya-bean peroxidase (SBP). Fourier transform infrared spectra of the surfaces show that protein is retained on the surface after boiling in sodium dodecyl sulphate and sodium hydroxide, which is indicative of covalent attachment. The activity of SBP on the treated surfaces remains high in comparison with SBP attached to control surfaces over the course of 11 days. Surface plasmon resonance was used to show that the surface coverage of the attached protein is close to a monolayer. We describe the potential of the PIII treatment method to be used as a one-step dry process to create surfaces for large-scale protein micro- or nanopatterning. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Dehghani, Fariba %A Annabi, Nasim %A Valtchev, Peter %A Mithieux, Suzanne M %A Weiss, Anthony S %A Kazarian, Sergei G %A Tay, Feng H %T Effect of dense gas CO2 on the coacervation of elastin. %B Biomacromolecules %D 2008 %C United States %I American Chemical Society %V 9 %N 4 %P 1100-1105 %@ 1526-4602 %X In this study for the first time the effect of high-pressure CO2 on the coacervation of alpha-elastin was investigated using analytical techniques including light spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging and circular dichroism (CD) spectroscopy. The coacervation behavior of alpha-elastin, a protein biopolymer, was determined at temperatures below 40 degrees C and pressures lower than 180 bar. At these conditions elevated pressures did not disrupt the ability of alpha-elastin to coacervate. It was feasible to monitor the presence of amide I, II, and III bands for alpha-elastin at high-pressure CO2 using ATR-FTIR imaging. At a constant temperature the peak absorption was substantially enhanced by increasing the pressure of the system. CD analysis demonstrated the preservation of secondary structure attributes of alpha-elastin exposed to dense gas CO2 at the pressure range investigated in this study. The lower critical solution temperature of alpha-elastin was dramatically decreased from 37 to 16 degrees C when the CO2 pressure increased from 1 to 50 bar, without a significant change after that. Carbon dioxide at high pressures also impeded the reversible coacervation of alpha-elastin solution. These effects were predominantly associated with the lowered pH of the aqueous solution and maybe the interaction between CO2 and hydrophobic domains of alpha-elastin. %Z FOR Codes: 1108 %0 Journal Article %~ PubMed %A Tu, Yidong %A Weiss, Anthony S %T Glycosaminoglycan-mediated coacervation of tropoelastin abolishes the critical concentration, accelerates coacervate formation, and facilitates spherule fusion: implications for tropoelastin microassembly. %B Biomacromolecules %D 2008 %C United States %I American Chemical Society %V 9 %N 7 %P 1739-44 %@ 1526-4602 %X Elastogenesis and elastin repair depend on the secretion of tropoelastin from the cell, yet cellular production is low in the many biological systems that have been studied. To address the apparent paradox of a paucity of tropoelastin for cell surface microassembly, we examined the effects of the glycosaminoglycans heparin, heparan sulfate, and chondroitin sulfate B, on tropoelastin aggregate formation through coacervation. We found a significant effect, particularly of heparin, on the minimum or critical concentration of tropoelastin, which was required for microassembly, lowering critical concentration to a point that it was no longer detectable. The assemblies resulted in protein droplet formation that was visually indistinguishable from the spherules that typify coacervation. The spherules readily coalesced in the presence of heparin and higher concentrations of tropoelastin, resulting in an almost continuous layer of coacervated tropoelastin. Four stages of droplet behavior were observed: early droplet formation, approximately 6 mum droplet formation, and fusion of droplets followed by the formation of a coalesced layer. We conclude that glycosaminoglycans in the extracellular matrix have the capacity to promote coacervation at low concentrations of tropoelastin. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Taddese, Samuel %A Weiss, Anthony S %A Neubert, Reinhard H H %A Schmelzer, Christian E H %T Mapping of macrophage elastase cleavage sites in insoluble human skin elastin. %B Matrix biology : journal of the International Society for Matrix Biology %D 2008 %C Netherlands %I Elsevier BV %V 27 %N %P 420-8 %@ 0945-053X %X Macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) and is active against multiple extracellular protein substrates such as elastin. Its effect on elastin is central to emphysema in the lung and photoaging of skin. Its expression in the skin increases on photodamaged skin and upon aging. Detecting and characterizing peptides cleaved in elastin, therefore, helps to understand such degradative disease processes in the skin and is also needed to assist in the rational design of agents that specifically inhibit the degradation. In this study, cleavage sites of MMP-12 in human skin elastin were extensively investigated. The peptides formed as a result of cleavages by this enzyme in the human skin elastin were characterized using mass spectrometry. A total of 41 peptides ranging from 4 to 41 amino acids were identified and 36 cleavage sites were determined. Amino acids encoded by exons 5, 6, 26, 28-31 were particularly susceptible to cleavages by MMP-12 and none or very few cleavages were detected from domains encoded by the remaining exons. The amino acid preferences of the different subsites on the catalytic domain of MMP-12 were analyzed. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Muiznieks, Lisa D %A Weiss, Anthony S %T Flexibility in the solution structure of human tropoelastin. %B Biochemistry %D 2007 %C United States %I American Chemical Society %V 46 %N 27 %P 8196-8205 %@ 0006-2960 %X We investigated the flexibility of full-length tropoelastin in solution by using far- and near-ultraviolet circular dichroism (UV CD) and fluorescence spectroscopy to probe for structural flexibility and residue mobility within secondary and tertiary features of the monomer. Fluorescence spectroscopy revealed the presence of exposed hydrophobicity through the binding of the hydrophobic probe 4,4''-dianilino-1,1''-binaphthyl-5,5''-disulfonate (bis-ANS), which demonstrates that hydrophobic regions form clusters and are not confined to a molecular core. Near-UV CD indicated substantial mobility of aromatic residues. Structural prediction programs (PONDR, DisEMBL, and Globplot version 2.0) estimated 75 +/- 2% disorder in the tertiary structure of tropoelastin on the basis of primary sequence information. A single-site substitution of Trp for Gln (Q513W) at the tropoelastin domain 25-26 interface facilitated fluorescence spectroscopy for revealing that this region is exposed to solvent. Polarization anisotropy demonstrated substantial flexibility of W513 and little change upon denaturation of the monomer with guanidine hydrochloride. Comparable movement was found for native sequence aromatic residues in the presence of glycosaminoglycans and trifluoroethanol. These data prove the intrinsic flexibility of specific residues and adjacent sequences in any native conformation(s) they may take. This study is the first characterization of the level of mobility in defined regions of the full-length tropoelastin monomer and provides direct evidence for regions of flexible structure in tropoelastin. %Z FOR Codes: %0 Journal Article %~ Isi %A Clarke, AW %A Foster, TJ %A Keane, FM %A Weiss, AS %T The N-terminal a domain of Staphylococcus aureus fibronectin-binding protein a binds to tropoelastin %B BIOCHEMISTRY %D 2007 %C United States %I American Chemical Society %V 46 %N 24 %P 7226-7232 %@ 0006-2960 %X %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Riley, Lisa G %A Roufogalis, Basil D %A Li, George Q %A Weiss, Anthony S %T A radioassay for synaptic core complex assembly: Screening of herbal extracts for effectors. %B Analytical biochemistry %D 2006 %C United States %I Academic Press %V 357 %N %P 50-7 %@ 0003-2697 %X Synaptic core complex formation between syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) on the plasma membrane and synaptobrevin on the vesicle membrane is responsible for membrane fusion and neurotransmitter release. A radiolabeled protein binding assay for synaptic core complex formation was developed. The components of this assay included recombinant glutathione S-transferase (GST)-syntaxin immobilized on glutathione agarose beads, SNAP25 and (125)I-labeled synaptobrevin. Reactions were performed in tubes containing filter inserts to facilitate removal of unbound protein. The radiolabeled protein bound was then quantified by gamma counter. A K(d) of 1.6 microM was determined for the GST-syntaxin/SNAP25/synaptobrevin complex, and a K(d) of 12 microM was determined for the GST-syntaxin/synaptobrevin complex. The assay was used to screen 14 herbal extracts for effectors of core complex formation. Herbs traditionally used to treat neurological conditions such as depression, anxiety, and stress were chosen. A Hypericum perforatum extract was found to have a nonspecific effect via protein complexation, whereas an Albizzia julibrissin extract was found to reduce the level of core complex formation. The assay was used to further investigate the effect of the A. julibrissin extract. The discovery of an inhibitor of core complex formation demonstrates the efficacy of the assay in screening natural products for substances that affect core complex formation. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Nicoloff, George %A Weiss, Antony S %A Iotova, Violeta %A Tzaneva, Valentina %A Petrova, Chayka %A Domuschieva, Nadja %A Nikolov, Asparuh %A Tzvetanov, Plamen %A Christova, Petkana %T Abnormal levels of serum antielastin antibodies in children with diabetes mellitus type 1. %B Journal of investigative medicine : the official publication of the American Federation for Clinical Research %D 2006 %C CANADA, ONTARIO %I B C DECKER INC %V 54 %N 8 %P 461-7 %@ 1081-5589 %X Antibodies to alpha-elastin (elastin breakdown product) and elastin sequences devoid of cross-linked regions (linear elastin) are found in the serum of all human subjects and correlate with their respective serum peptide levels. The aim of this study was to determine if the serum level of antielastin antibodies (AEAbs) differs between type 1 diabetic children and nondiabetic children. Enzyme-linked immunosorbent assay was used to measure the levels of immunoglobulin (Ig)G and IgM AEAbs in the sera of 45 diabetic children (mean age 12.8 +/- 3.2 years, diabetes duration 5.3 +/- 3.6 years). Twenty-two children presented with vascular complications (group 1), whereas 23 displayed no vascular complications (group 2). The controls were 18 healthy children (mean age 11.9 +/- 2.3 years). Diabetic patients showed statistically significant higher levels of IgM alpha-AEAbs (0.82 +/- 0.26 vs 0.61 +/- 0.14, p = .0013) than the control group. In group 1, alpha-AEAbs showed statistically significant higher level than controls: IgG (0.86 +/- 0.42 vs 0.59 +/- 0.12; p = .0109) and IgM (0.88 +/- 0.24 vs 0.61 +/- 0.14; p = .0001). IgM antilinear elastin antibodies (ALEAbs) in group 1 were significantly lower than in controls (0.462 +/- 0.191 vs 0.652 +/- 0.127; p = .0009). IgG alpha-AEAbs showed correlation with microalbuminuria (r = -.26; p = .05) and IgM ALEAbs correlated with microalbuminuria (r = -.32; p = .035). IgG alpha-AEAbs correlated with neuropathy (r = -.32; p = .035). Group 1 patients displayed a correlation between IgG ALEAbs and retinopathy (r = -.48; p = .023) and IgM ALEAbs and microalbuminuria (r = .52; p = .014). Levels of AEAbs and ALEAbs can serve as immunologic markers of the extent of elastin degradation. These markers may provide a tool to study elastin metabolism and a potential clinical role for AEAbs in the pathogenesis and development of vascular complications in diabetic children. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Lemire, Joan M %A Patis, Carrie %A Gordon, Leslie B %A Sandy, John D %A Toole, Bryan P %A Weiss, Anthony S %T Aggrecan expression is substantially and abnormally upregulated in Hutchinson-Gilford Progeria Syndrome dermal fibroblasts. %B Mechanisms of ageing and development %D 2006 %C Ireland %I Elsevier Ireland Ltd %V 127 %N 8 %P 660-9 %@ 0047-6374 %X Hutchinson-Gilford Progeria syndrome (HGPS) is a rare genetic disorder that displays features of segmental aging. It is manifested predominantly in connective tissue, with most prominent histological changes occurring in the skin, cartilage, bone and cardiovascular tissues. Detailed quantitative real time reverse-transcription polymerase chain reaction studies confirmed the previous observation that platelet-derived growth factor A-chain transcripts are consistently elevated 11+/-2- to 13+/-2-fold in two HGPS dermal fibroblast lines compared with age-matched controls. Furthermore, we identified two additional genes with substantially altered transcript levels. Nucleotide pyrophosphatase transcription was virtually shut down with decreased expression of 13+/-3- to 59+/-3-fold in HGPS, whereas aggrecan mRNA was elevated to 24+/-5 times to 41+/-4 times that of chronologically age-matched controls. Aggrecan, normally a component of cartilage and not always detectable in normal fibroblasts cultures, was secreted by HGPS fibroblast lines and was produced as a proteoglycan. This demonstrates that elevated aggrecan expression and its secretion are aberrant features of HGPS. We conclude that HGPS cells can display massively altered transcript levels leading to the secretion of inappropriate protein species. %Z FOR Codes: 110102 111601 %0 Journal Article %~ PubMed %A Dyksterhuis, L B %A Baldock, C %A Lammie, D %A Wess, T J %A Weiss, A S %T Domains 17-27 of tropoelastin contain key regions of contact for coacervation and contain an unusual turn-containing crosslinking domain. %B Matrix biology : journal of the International Society for Matrix Biology %D 2006 %C Amsterdam %I ELSEVIER SCIENCE BV %V 26 %N %P 125-35 %@ 0945-053X %X The central region of tropoelastin including domains 19-25 of human tropoelastin forms a hot-spot for contacts during the inter-molecular association of tropoelastin by coacervation [Wise, S.G., Mithieux, S.M., Raftery, M.J. and Weiss, A.S (2005). "Specificity in the coacervation of tropoelastin: solvent exposed lysines." Journal of Structural Biology 149: 273-81.]. We explored the physical properties of this central region using a sub-fragment bordered by domains 17-27 of human tropoelastin (SHEL 17-27) and identified the intra- and inter-molecular contacts it forms during coacervation. A homobifunctional amine reactive crosslinker (with a maximum reach of 11 A, corresponding to approximately 7 residues in an extended polypeptide chain) was used to capture these contacts and crosslinked regions were identified after protease cleavage and mass spectrometry (MS) with MS/MS verification. An intermolecular crosslink formed between the lysines at positions 353 of each strand of tropoelastin at the lowest of crosslinker concentrations and was observed in all samples tested, suggesting that this residue forms an important initial contact during coacervation. At higher crosslinker concentrations, residues K425 and K437 showed the highest levels of involvement in crosslinks. An intramolecular crosslink between these K425 and K437, separated by 11 residues, indicated that a structural bend must serve to bring these residues into close proximity. These studies were complemented by small angle X-ray scattering studies that confirmed a bend in this important subfragment of the tropoelastin molecule. %Z FOR Codes: 110106 %0 Journal Article %~ PubMed %A Clarke, Adam %A Arnspang, Eva %A Mithieux, Suzanne %A Korkmaz, Emine %A Braet, Filip %A Weiss, Anthony %T Tropoelastin Massively Associates during Coacervation To Form Quantized Protein Spheres. %B Biochemistry %D 2006 %C 1155 16Th St, Nw, Wa %I Amer Chemical Soc %V 45 %N 33 %P 9989-9996 %@ 0006-2960 %X Tropoelastin, the precursor of elastin, undergoes a rapid monomer to multimer association in an inverse temperature transition. This association culminates in the rapid formation of stable, optically distinct droplets of tropoelastin. Light scattering and microscope measurements reveal that these droplets are 2-6 mum in diameter. Scanning electron microscopy confirms that the droplets are spherical. Three-dimensional confocal image stacks based on the autofluorescence of tropoelastin reveal that droplets are loaded with hydrated tropoelastin. Droplets are viable intermediates in synthetic elastin macroassembly. Dense clusters of aggregated droplets and partially formed fibers develop when droplets are incubated in the presence of a lysyl oxidase. Lysine-reacting chemical and enzyme-assisted cross-linking conditions generate cross-linked beads due to interactions between multiple, surface-exposed lysine epsilon-amino groups. Droplets represent an efficient mechanism for the bolus delivery during elastogenesis of quantized packages of preaccreted tropoelastin. %Z FOR Codes: 110106