RESUMO
BACKGROUND: Myocardial infarction (MI) is among the leading causes of death worldwide. Following MI, necrotic cardiomyocytes are replaced by a stiff collagen-rich scar. Compared to collagen, the extracellular matrix protein elastin has high elasticity and may have more favorable properties within the cardiac scar. We sought to improve post-MI healing by introducing tropoelastin, the soluble subunit of elastin, to alter scar mechanics early after MI. METHODS AND RESULTS: We developed an ultrasound-guided direct intramyocardial injection method to administer tropoelastin directly into the left ventricular anterior wall of rats subjected to induced MI. Experimental groups included shams and infarcted rats injected with either PBS vehicle control or tropoelastin. Compared to vehicle treated controls, echocardiography assessments showed tropoelastin significantly improved left ventricular ejection fraction (64.7±4.4% versus 46.0±3.1% control) and reduced left ventricular dyssynchrony (11.4±3.5 ms versus 31.1±5.8 ms control) 28 days post-MI. Additionally, tropoelastin reduced post-MI scar size (8.9±1.5% versus 20.9±2.7% control) and increased scar elastin (22±5.8% versus 6.2±1.5% control) as determined by histological assessments. RNA sequencing (RNAseq) analyses of rat infarcts showed that tropoelastin injection increased genes associated with elastic fiber formation 7 days post-MI and reduced genes associated with immune response 11 days post-MI. To show translational relevance, we performed immunohistochemical analyses on human ischemic heart disease cardiac samples and showed an increase in tropoelastin within fibrotic areas. Using RNA-seq we also demonstrated the tropoelastin gene ELN is upregulated in human ischemic heart disease and during human cardiac fibroblast-myofibroblast differentiation. Furthermore, we showed by immunocytochemistry that human cardiac fibroblast synthesize increased elastin in direct response to tropoelastin treatment. CONCLUSIONS: We demonstrate for the first time that purified human tropoelastin can significantly repair the infarcted heart in a rodent model of MI and that human cardiac fibroblast synthesize elastin. Since human cardiac fibroblasts are primarily responsible for post-MI scar synthesis, our findings suggest exciting future clinical translation options designed to therapeutically manipulate this synthesis.
Assuntos
Infarto do Miocárdio , Miocárdio , Humanos , Ratos , Animais , Miocárdio/metabolismo , Elastina/metabolismo , Tropoelastina/genética , Tropoelastina/metabolismo , Cicatriz , Volume Sistólico , Função Ventricular Esquerda , Miócitos Cardíacos/metabolismo , Colágeno/metabolismo , Remodelação VentricularRESUMO
We challenge the conventional designation of structural matrix proteins primarily as supporting scaffolds for resident cells. The extracellular matrix protein tropoelastin is classically regarded as a structural component that confers mechanical strength and resilience to tissues subject to repetitive elastic deformation. Here we describe how tropoelastin inherently induces a range of biological responses, even in cells not typically associated with elastic tissues and in a manner unexpected of typical substrate-dependent matrix proteins. We show that tropoelastin alone drives mesenchymal stem cell (MSC) proliferation and phenotypic maintenance, akin to the synergistic effects of potent growth factors such as insulin-like growth factor 1 and basic fibroblast growth factor. In addition, tropoelastin functionally surpasses these growth factors, as well as fibronectin, in allowing substantial media serum reduction without loss of proliferative potential. We further demonstrate that tropoelastin elicits strong mitogenic and cell-attractive responses, both as an immobilized substrate and as a soluble additive, via direct interactions with cell surface integrins αvß3 and αvß5. This duality of action converges the long-held mechanistic dichotomy between adhesive matrix proteins and soluble growth factors and uncovers the powerful, untapped potential of tropoelastin for clinical MSC expansion and therapeutic MSC recruitment. We propose that the potent, growth factor-like mitogenic and motogenic abilities of tropoelastin are biologically rooted in the need for rapid stem cell homing and proliferation during early development and/or wound repair.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Tropoelastina/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos , Fator de Crescimento Insulin-Like I/efeitos dos fármacos , Integrina alfaVbeta3 , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores de Vitronectina , CicatrizaçãoRESUMO
The discovery that cells secrete extracellular vesicles (EVs), which carry a variety of regulatory proteins, nucleic acids, and lipids, has shed light on the sophisticated manner by which cells can communicate and accordingly function. The bioactivity of EVs is not only defined by their internal content, but also through their surface associated molecules, and the linked downstream signaling effects they elicit in target cells. The extracellular matrix (ECM) contains signaling and structural molecules that are central to tissue maintenance and repair. Recently, a subset of EVs residing within the extracellular matrix has been identified. Although some roles have been proposed for matrix-bound vesicles, their role as signaling molecules within the ECM is yet to be explored. Given the close association of EVs and the ECM, it is not surprising that EVs partly mediate repair and regeneration by modulating matrix deposition and degradation through their cellular targets. This review addresses unique EV features that allow them to interact with and navigate through the ECM, describes how their release and content is influenced by the ECM, and emphasizes the emerging role of stem-cell derived EVs in tissue repair and regeneration through their matrix-modulating properties.
Assuntos
Vesículas Extracelulares , Ácidos Nucleicos , Transporte Biológico , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Ácidos Nucleicos/metabolismo , Células-Tronco/metabolismoRESUMO
Tropoelastin is the highly flexible monomer subunit of elastin, required for the resilience of the extracellular matrix in elastic tissues. To elicit biological signaling, multiple sites on tropoelastin bind to cell surface integrins in a poorly understood multifactorial process. We constructed a full atomistic molecular model of the interactions between tropoelastin and integrin αvß3 using ensemble-based computational methodologies. Conformational changes of integrin αvß3 associated with outside-in signaling were more frequently facilitated in an ensemble in which tropoelastin bound the integrin's α1 helix rather than the upstream canonical binding site. Our findings support a model of fuzzy binding, whereby many tropoelastin conformations and defined sites cooperatively interact with multiple αvß3 regions. This model explains prior experimental binding to distinct tropoelastin regions, domains 17 and 36, and points to the cooperative participation of domain 20. Our study highlights the utility of ensemble-based approaches in helping to understand the interactive mechanisms of functionally significant flexible proteins.
Assuntos
Integrina alfaVbeta3 , Tropoelastina , Sítios de Ligação , Elastina , Matriz Extracelular , HumanosRESUMO
Protein folding poses unique challenges for large, disordered proteins due to the low resolution of structural data accessible in experiment and on the basis of short time scales and limited sampling attainable in computation. Such molecules are uniquely suited to accelerated-sampling molecular dynamics algorithms due to a flat-energy landscape. We apply these methods to report here the folded structure in water from a fully extended chain of tropoelastin, a 698-amino acid molecular precursor to elastic fibers that confer elasticity and recoil to tissues, finding good agreement with experimental data. We then study a series of artificial and disease-related mutations, yielding molecular mechanisms to explain structural differences and variation in hierarchical assembly observed in experiment. The present model builds a framework for studying assembly and disease and yields critical insight into molecular mechanisms behind these processes. These results suggest that proteins with disordered regions are suitable candidates for characterization by this approach.
Assuntos
Simulação de Dinâmica Molecular , Mutação , Tropoelastina/química , Humanos , Tropoelastina/genética , Tropoelastina/metabolismoRESUMO
Cardiovascular disorders are a healthcare problem in today's society. The clinically available synthetic vascular grafts are thrombogenic and could induce intimal hyperplasia. Rapid endothelialization and matched mechanical properties are two major requirements to be considered when designing functional vascular grafts. Herein, an electrospun tubular fibrous (eTF) scaffold was biofunctionalized with tropoelastin at the luminal surface. The luminal surface functionalization was confirmed by an increase of the zeta potential and by the insertion of NH2 groups. Tropoelastin was immobilized via its -NH2 or -COOH groups at the activated or aminolysed eTF scaffolds, respectively, to study the effect of exposed functional groups on human endothelial cells (ECs) behavior. Tensile properties demonstrated that functionalized eTF scaffolds presented strength and stiffness within the range of those of native blood vessels. Tropoelastin immobilized on activated eTF scaffolds promoted higher metabolic activity and proliferation of ECs, whereas when immobilized on aminolysed eTF scaffolds, significantly higher protein synthesis was observed. These biofunctional eTF scaffolds are a promising small-diameter vascular graft that promote rapid endothelialization and have compatible mechanical properties.
Assuntos
Tropoelastina , Enxerto Vascular , Prótese Vascular , Células Endoteliais , Humanos , Engenharia Tecidual , Alicerces TeciduaisRESUMO
BACKGROUND: Elastin is an essential component of the dermis, providing skin with elasticity and integrity. Elastin and other dermal components are gradually lost through aging, sun damage, and following injury, highlighting a need to replace these components to repair the skin. Tropoelastin (TE) in monomeric form was previously shown to be utilized as a substrate by dermal fibroblasts during the production of elastin fibers in vitro. OBJECTIVE: To analyze coaccumulation of elastin and collagen and gene expression of biomarkers associated with elastin production, examine the ex vivo effects of recombinant human TE (rhTE) and hyaluronic acid (HA) on epidermal and dermal structures, and evaluate the in vivo response following intradermal injections of rhTE and HA. METHODS: Human dermal fibroblasts and 3-D skin patch models were cultured for in vitro analysis. Ex vivo analysis was performed using skin explants. In vivo studies were done in 6-week-old male CD Hairless rats. Different formulations of rhTE, soluble or crosslinked using derivatized HA (dHA), were tested and analyzed. RESULTS: rhTE in monomeric form was utilized as a substrate by dermal fibroblasts during the production of branched elastin and fibrous collagen networks in vitro. Formulations of rhTE crosslinked with dHA demonstrated increased expression of hyaluronic acid synthase 1 and ex vivo results revealed increased moisture content and glycosaminoglycan (GAG) deposition versus dermal filler control. Intradermal rhTEâdHA injection produced colocalized humanârat elastin fibers in vivo. CONCLUSIONS: These results suggest that the novel rhTEâdHA matrix is an attractive material to support skin tissue repair.J Drugs Dermatol. 2020;19(12): doi:10.36849/JDD.2020.5375.
Assuntos
Preenchedores Dérmicos/administração & dosagem , Matriz Extracelular/efeitos dos fármacos , Envelhecimento da Pele/efeitos dos fármacos , Pele/efeitos dos fármacos , Tropoelastina/administração & dosagem , Animais , Linhagem Celular , Colágeno/análise , Colágeno/metabolismo , Técnicas Cosméticas , Implantes de Medicamento , Elastina/análise , Elastina/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fibroblastos , Glicosaminoglicanos/análise , Glicosaminoglicanos/metabolismo , Humanos , Ácido Hialurônico/administração & dosagem , Injeções Intradérmicas , Masculino , Pessoa de Meia-Idade , Modelos Animais , Ratos , Proteínas Recombinantes/administração & dosagem , Pele/química , Pele/citologia , Pele/metabolismo , Técnicas de Cultura de TecidosRESUMO
Alpha-1 antitrypsin (AAT) deficiency-related emphysema is the fourth leading indication for lung transplant. Chymotrypsin-like elastase 1 (Cela1) is a digestive protease that is expressed during lung development in association with regions of elastin remodeling, exhibits stretch-dependent expression during lung regeneration, and binds lung elastin in a stretch-dependent manner. AAT covalently neutralizes Cela1 in vitro. We sought to determine the role of Cela1 in postnatal lung physiology, whether it interacted with AAT in vivo, and to detect any effects it may have in the context of AAT deficiency. The lungs of Cela1-/- mice had aberrant lung elastin structure and higher elastance as assessed with the flexiVent system. On the basis of in situ zymography with ex vivo lung stretch, Cela1 was solely responsible for stretch-inducible lung elastase activity. By mass spectrometry, Cela1 degraded mature elastin similarly to pancreatic elastase. Cela1 promoter and protein sequences were phylogenetically distinct in the placental mammal lineage, suggesting an adaptive role for lung-expressed Cela1 in this clade. A 6-week antisense oligonucleotide mouse model of AAT deficiency resulted in emphysema with increased Cela1 mRNA and reduction of approximately 70 kD Cela1, consistent with covalent binding of Cela1 by AAT. Cela1-/- mice were completely protected against emphysema in this model. Cela1 was increased in human AAT-deficient emphysema. Cela1 is important in physiologic and pathologic stretch-dependent remodeling processes in the postnatal lung. AAT is an important regulator of this process. Our findings provide proof of concept for the development of anti-Cela1 therapies to prevent and/or treat AAT-deficient emphysema.
Assuntos
Enfisema/genética , Regulação Enzimológica da Expressão Gênica/genética , Elastase Pancreática/metabolismo , alfa 1-Antitripsina/genética , Animais , Fenômenos Biomecânicos , Elastina/metabolismo , Fibroblastos/metabolismo , Humanos , Pulmão/crescimento & desenvolvimento , Camundongos Knockout , Elastase Pancreática/genéticaRESUMO
PDMS is widely used for prosthetic device manufacture. Conventional ion implantation is not a suitable treatment to enhance the biocompatibility of poly dimethyl siloxane (PDMS) due to its propensity to generate a brittle silicon oxide surface layer which cracks and delaminates. To overcome this limitation, we have developed new plasma based processes to balance the etching of carbon with implantation of carbon from the plasma source. When this carbon was implanted from the plasma phase it resulted in a surface that was structurally similar and intermixed with the underlying PDMS material and not susceptible to delamination. The enrichment in surface carbon allowed the formation of carbon based radicals that are not present in conventional plasma ion immersion implantation (PIII) treated PDMS. This imparts the PDMS surfaces with covalent protein binding capacity that is not observed on PIII treated PDMS. The change in surface energy preserved the function of bound biomolecules and enhanced the attachment of MG63 osteosarcoma cells compared to the native surface. The attached cells, an osteoblast interaction model, showed increased spreading on the treated over untreated surfaces. The carbon-dependency for these beneficial covalent protein and cell linkage properties was tested by incorporating carbon from a different source. To this end, a second surface was produced where carbon etching was balanced against implantation from a thin carbon-based polymer coating. This had similar protein and cell-binding properties to the surfaces generated with carbon inclusion in the plasma phase, thus highlighting the importance of balancing carbon etching and deposition. Additionally, the two effects of protein linkage and bioactivity could be combined where the cell response was further enhanced by covalently tethering a biomolecule coating, as exemplified here with the cell adhesive protein tropoelastin. Providing a balanced carbon source in the plasma phase is applicable to prosthetic device fabrication as illustrated using a 3-dimensional PDMS balloon prosthesis for spinal implant applications. Consequently, this study lays the groundwork for effective treatments of PDMS to selectively recruit cells to implantable PDMS fabricated biodevices.
Assuntos
Adesão Celular , Materiais Revestidos Biocompatíveis/química , Dimetilpolisiloxanos/química , Proteínas Imobilizadas , Linhagem Celular Tumoral , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Osteossarcoma , Próteses e Implantes , Ligação Proteica , Propriedades de SuperfícieRESUMO
Tropoelastin is an extracellular matrix protein that assembles into elastic fibers that provide elasticity and strength to vertebrate tissues. Although the contributions of specific tropoelastin regions during each stage of elastogenesis are still not fully understood, studies predominantly recognize the central hinge/bridge and C-terminal foot as the major participants in tropoelastin assembly, with a number of interactions mediated by the abundant positively charged residues within these regions. However, much less is known about the importance of the rarely occurring negatively charged residues and the N-terminal coil region in tropoelastin assembly. The sole negatively charged residue in the first half of human tropoelastin is aspartate 72. In contrast, the same region comprises 17 positively charged residues. We mutated this aspartate residue to alanine and assessed the elastogenic capacity of this novel construct. We found that D72A tropoelastin has a decreased propensity for initial self-association, and it cross-links aberrantly into denser, less porous hydrogels with reduced swelling properties. Although the mutant can bind cells normally, it does not form elastic fibers with human dermal fibroblasts and forms fewer atypical fibers with human retinal pigmented epithelial cells. This impaired functionality is associated with conformational changes in the N-terminal region. Our results strongly point to the role of the Asp-72 site in stabilizing the N-terminal segment of human tropoelastin and the importance of this region in facilitating elastic fiber assembly.
Assuntos
Ácido Aspártico , Elasticidade , Tropoelastina/química , Tropoelastina/metabolismo , Fibroblastos/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pele/citologia , Tropoelastina/genéticaRESUMO
Tropoelastin protein monomers assemble to form elastin. Cellular integrin αVß3 binds RKRK at the C-terminal tail of tropoelastin. We probed cell interactions with tropoelastin by deleting the RKRK sequence to identify other cell-binding interactions within tropoelastin. We found a novel human dermal fibroblast attachment and spreading site on tropoelastin that is located centrally in the molecule. Inhibition studies demonstrated that this cell adhesion was not mediated by either elastin-binding protein or glycosaminoglycans. Cell interactions were divalent cation-dependent, indicating integrin dependence. Function-blocking monoclonal antibodies revealed that αV integrin(s) and integrin αVß5 specifically were critical for cell adhesion to this part of tropoelastin. These data reveal a common αV integrin-binding theme for tropoelastin: αVß3 at the C terminus and αVß5 at the central region of tropoelastin. Each αV region contributes to fibroblast attachment and spreading, but they differ in their effects on cytoskeletal assembly.
Assuntos
Fibroblastos/metabolismo , Receptores de Vitronectina/metabolismo , Tropoelastina/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Fibroblastos/citologia , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Receptores de Vitronectina/genética , Tropoelastina/genéticaRESUMO
Elastases have been widely studied because of their important uses as medicine and meat tenderizers. However, there are relatively few studies on marine elastases. Myroilysin, secreted by Myroides profundi D25 from deep-sea sediment, is a novel elastase. In this study, we examined the elastin degradation mechanism of myroilysin. When mixed with insoluble bovine elastin, myroilysin bound hydrophobically, suggesting that this elastase may interact with the hydrophobic domains of elastin. Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1' positions. Scanning electron microscopy (SEM) of cross-linked recombinant tropoelastin degraded by myroilysin showed preferential damages of spherules over cross-links, as expected for a hydrophobic preference. The degradation process of myroilysin on bovine elastin fibres was followed by light microscopy and SEM, revealing that degradation begins with the formation of crevices and cavities at the fibre surface, with these openings increasing in number and size until the fibre breaks into small pieces, which are subsequently fragmented. Our results are helpful for developing biotechnological applications for myroilysin.
Assuntos
Elastina/metabolismo , Flavobacteriaceae/enzimologia , Sedimentos Geológicos/microbiologia , Metaloproteases/metabolismo , Animais , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Metaloproteases/isolamento & purificação , Microscopia Eletrônica de Varredura , Tropoelastina/metabolismoRESUMO
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.
Assuntos
Comunicação Celular , Tecido Elástico/metabolismo , Tropoelastina/química , Tropoelastina/metabolismo , Adesão Celular , Células Cultivadas , Tecido Elástico/química , Tecido Elástico/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Hidrogéis , Microscopia Confocal , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Tamanho da Partícula , Estrutura Terciária de Proteína , Proteólise , Soluções , Relação Estrutura-Atividade , Temperatura , Tropoelastina/ultraestruturaRESUMO
Following penetrating injury of the skin, a highly orchestrated and overlapping sequence of events helps to facilitate wound resolution. Inflammation is a hallmark that is initiated early, but the reciprocal relationship between cells and matrix molecules that triggers and maintains inflammation is poorly appreciated. Elastin is enriched in the deep dermis of skin. We propose that deep tissue injury encompasses elastin damage, yielding solubilized elastin that triggers inflammation. As dermal fibroblasts dominate the deep dermis, this means that a direct interaction between elastin sequences and fibroblasts would reveal a proinflammatory signature. Tropoelastin was used as a surrogate for elastin sequences. Tropoelastin triggered fibroblast expression of the metalloelastase MMP-12, which is normally expressed by macrophages. MMP-12 expression increased 1056 ± 286-fold by 6 h and persisted for 24 h. Chemokine expression was more transient, as chemokine C-X-C motif ligand 8 (CXCL8), CXCL1, and CXCL5 transcripts increased 11.8 ± 2.6-, 10.2 ± 0.4-, and 8593 ± 996-fold, respectively, by 6-12 h and then decreased. Through the use of specific inhibitors and protein truncation, we found that transduction of the tropoelastin signal was mediated by the fibroblast elastin binding protein (EBP). In silico modeling using a predictive computational fibroblast model confirmed the up-regulation, and simulations revealed PKA as a key part of the signaling circuit. We tested this prediction with 1 µM PKA inhibitor H-89 and found that 2 h of exposure correspondingly reduced expression of MMP-12 (63.9±12.3%) and all chemokine markers, consistent with the levels seen with EBP inhibition, and validated PKA as a novel node and druggable target to ameliorate the proinflammatory state. A separate trigger that utilized C-terminal RKRK of tropoelastin reduced marker expression to 65.0-76.5% and suggests the parallel involvement of integrin αVß3. We propose that the solubilization of elastin as a result of dermal damage leads to rapid chemokine up-regulation by fibroblasts that is quenched when exposed elastin is removed by MMP-12.
Assuntos
Derme/citologia , Elastina/metabolismo , Fibroblastos/metabolismo , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibroblastos/citologia , Humanos , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tropoelastina/farmacologia , Cicatrização/efeitos dos fármacosRESUMO
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.
Assuntos
Elasticidade , Especificidade de Órgãos , Tropoelastina/química , Animais , Entropia , Humanos , Modelos Moleculares , Difração de Nêutrons , Conformação Proteica , Espalhamento a Baixo Ângulo , Soluções , Vertebrados/metabolismo , Difração de Raios XRESUMO
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.
Assuntos
Equipamentos e Provisões/efeitos adversos , Radicais Livres , Máquina Coração-Pulmão/efeitos adversos , Proteínas/química , Stents/efeitos adversos , Humanos , Conformação Proteica , Propriedades de SuperfícieRESUMO
Synthetic vascular grafts are commonly used in patients with severe occlusive arterial disease when autologous grafts are not an option. Commercially available synthetic grafts are confronted with challenging outcomes: they have a lower patency rate than autologous grafts and are currently unable to promote arterial regeneration. Polyglycerol sebacate (PGS), a non-toxic polymer with a tunable degradation profile, has shown promising results as a small-diameter vascular graft component that can support the formation of neoarteries. In this review, we first present an overview of the synthesis and modification of PGS followed by an examination of its mechanical properties. We then report on the performance, degradation, regeneration, and remodeling of PGS-based small-diameter vascular grafts, with a focus on efforts to reduce thrombosis, prevent dilation, and promote cellular residency and extracellular matrix regeneration that resembles the native artery in spatial distribution and organization. We also highlight recent advances in the incorporation of novel in situ cell sources for arterial regeneration and their potential application in PGS-based vascular grafts. Finally, we compare vascular grafts fabricated using PGS-based materials with other elastomeric alternatives.
Assuntos
Artérias , Glicerol , Polímeros , Humanos , Polímeros/farmacologia , Prótese Vascular , RegeneraçãoRESUMO
Cellular senescence leads to the functional decline of regenerative cells such as mesenchymal stromal/stem cells (MSCs), which gives rise to chronic conditions and contributes to poor cell therapy outcomes. Aging tissues are associated with extracellular matrix (ECM) dysregulation, including loss of elastin. However, the role of the ECM in modulating senescence is underexplored. In this work, it is shown that tropoelastin, the soluble elastin precursor, is not only a marker of young MSCs but also actively preserves cell fitness and delays senescence during replicative aging. MSCs briefly exposed to tropoelastin exhibit upregulation of proliferative genes and concurrent downregulation of senescence genes. The seno-protective benefits of tropoelastin persist during continuous, long-term MSC culture, and significantly extend the MSC replicative lifespan. Tropoelastin-expanded MSCs further maintain youth-associated phenotype and function compared to age-matched controls, including preserved clonogenic potential, minimal senescence-associated beta-galactosidase activity, maintained cell sizes, reduced expression of senescence markers, suppressed secretion of senescence-associated factors, and increased production of youth-associated proteins. This work points to the utility of exogenously-supplemented tropoelastin for manufacturing MSCs that robustly maintain regenerative potential with age. It further reveals the active role of classical structural ECM proteins in driving cellular age-associated fitness, potentially leading to future interventions for aging-related pathologies.
RESUMO
The development of small-diameter vascular grafts requires testing in large animal models before advancing to clinical trials. Vascular graft interposition implantation in sheep carotid arteries (CAs) is the most widely used model, but ovine CAs are prone to severe spasm following surgical manipulation, potentially impairing graft performance assessment. There is paucity in the literature on reducing sheep CA spasm using effective vasodilator therapeutic protocols. In this study, four healthy Merino cross White Suffolk wethers (1-2 years, 52.1 ± 0.8 kg) underwent CT angiography and CA graft surgery. CT angiography using iodinated contrast agent was performed with innominate artery access through the CA or ascending aortic arch access through the femoral artery. Sheep then underwent right CA sham surgery or left CA vascular graft implantation. A variety of vasodilators, including papaverine, sodium nitroprusside, verapamil, and their combination, were tested for preventing or treating CA spasms intraoperatively. Blood flow was reassessed immediately after CA surgery using CT angiography. The results showed that innominate artery access through the CA for CT angiography in sheep induced presurgical CA spasm with reduced arterial flow. Conversely, ascending aortic arch access through the femoral artery for CT angiography did not cause CA spasm and maintained arterial flow. During CA graft surgery, surgical trauma induced CA spasm, which was prevented by localized intra-arterial administration of vasodilators papaverine hydrochloride and verapamil before significant surgical manipulation.
Assuntos
Artérias Carótidas , Angiografia por Tomografia Computadorizada , Animais , Ovinos , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/efeitos dos fármacos , Vasodilatadores/farmacologia , Enxerto Vascular/métodos , FemininoRESUMO
Wound healing is facilitated by biomaterials-based grafts and substantially impacted by orchestrated inflammatory responses that are essential to the normal repair process. Tropoelastin (TE) based materials are known to shorten the period for wound repair but the mechanism of anti-inflammatory performance is not known. To explore this, we compared the performance of the gold standard Integra Dermal Regeneration Template (Integra), polyglycerol sebacate (PGS), and TE blended with PGS, in a murine full-thickness cutaneous wound healing study. Systemically, blending with TE favorably increased the F4/80+ macrophage population by day 7 in the spleen and contemporaneously induced elevated plasma levels of anti-inflammatory IL-10. In contrast, the PGS graft without TE prompted prolonged inflammation, as evidenced by splenomegaly and greater splenic granulocyte and monocyte fractions at day 14. Locally, the inclusion of TE in the graft led to increased anti-inflammatory M2 macrophages and CD4+T cells at the wound site, and a rise in Foxp3+ regulatory T cells in the wound bed by day 7. We conclude that the TE-incorporated skin graft delivers a pro-healing environment by modulating systemic and local tissue responses. STATEMENT OF SIGNIFICANCE: Tropoelastin (TE) has shown significant benefits in promoting the repair and regeneration of damaged human tissues. In this study, we show that TE promotes an anti-inflammatory environment that facilitates cutaneous wound healing. In a mouse model, we find that inserting a TE-containing material into a full-thickness wound results in defined, pro-healing local and systemic tissue responses. These findings advance our understanding of TE's restorative value in tissue engineering and regenerative medicine, and pave the way for clinical applications.