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Sample records for extracellular matrix rigidity

  1. Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

    PubMed Central

    El-Hamidi, Hamid; Celli, Jonathan P.

    2014-01-01

    The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(ω). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic

  2. Extracellular matrix rigidity governs smooth muscle cell motility in a biphasic fashion.

    PubMed

    Peyton, Shelly R; Putnam, Andrew J

    2005-07-01

    Increasing evidence suggests that mechanical cues inherent to the extracellular matrix (ECM) may be equally as critical as its chemical identity in regulating cell behavior. We hypothesized that the mechanical properties of the ECM directly regulate the motility of vascular smooth muscle cells (SMCs) and tested this hypothesis using polyacrylamide substrates with tunable mechanical properties. Quantification of the migration speed on uniformly compliant hydrogels spanning a range of stiffnesses (Young's moduli values from 1.0 to 308 kPa for acrylamide/bisacrylamide ratios between 5/0.1% and 15/1.2%, respectively) revealed a biphasic dependence on substrate compliance, suggesting the existence of an optimal substrate stiffness capable of supporting maximal migration. The value of this optimal stiffness shifted depending on the concentration of ECM protein covalently attached to the substrate. Specifically, on substrates presenting a theoretical density of 0.8 microg/cm(2) fibronectin, the maximum speed of 0.74 +/- 0.09 microm/min was achieved on a 51.9 kPa gel; on substrates presenting a theoretical density of 8.0 microg/cm(2) fibronectin, the maximum speed of 0.72 +/- 0.06 microm/min occurred on a softer 21.6 kPa gel. Pre-treatment of cells with Y27632, an inhibitor of the Rho/Rho-kinase (ROCK) pathway, reduced these observed maxima to values comparable to those on non-optimal stiffnesses. In parallel, quantification of TritonX-insoluble vinculin via Western blotting, coupled with qualitative fluorescent microscopy, revealed that the formation of focal adhesions and actin stress fibers also depends on ECM stiffness. Combined, these data suggest that the mechanical properties of the underlying ECM regulate Rho-mediated contractility in SMCs by disrupting a presumptive cell-ECM force balance, which in turn regulates cytoskeletal assembly and ultimately, cell migration. (c) 2004 Wiley-Liss, Inc.

  3. Extracellular matrix rigidity modulates neuroblastoma cell differentiation and N-myc expression

    PubMed Central

    2010-01-01

    Neuroblastoma is a pediatric malignancy characterized by tremendous clinical heterogeneity, in which some tumors are extremely aggressive while others spontaneously differentiate into benign forms. Because the degree of differentiation correlates with prognosis, and because differentiating agents such as retinoic acid (RA) have proven to decrease mortality, much effort has been devoted to identifying critical regulators of neuroblastoma differentiation in the cellular microenvironment, including cues encoded in the extracellular matrix (ECM). While signaling between tumor cells and the ECM is classically regarded to be based purely on biochemical recognition of ECM ligands by specific cellular receptors, a number of recent studies have made it increasingly clear that the biophysical properties of the ECM may also play an important role in this cross-talk. Given that RA-mediated neuroblastoma differentiation is accompanied by profound changes in cell morphology and neurite extension, both of which presumably rely upon mechanotransductive signaling systems, it occurred to us that mechanical cues from the ECM might also influence RA-mediated differentiation, which in turn might regulate clinically-relevant aspects of neuroblastoma biology. In this study, we tested this hypothesis by subjecting a series of neuroblastoma culture models to ECM microenvironments of varying mechanical stiffness and examined the regulatory role of ECM stiffness in proliferation, differentiation, and expression of tumor markers. We find that increasing ECM stiffness enhances neuritogenesis and suppresses cell proliferation. Remarkably, increasing ECM stiffness also reduces expression of N-Myc, a transcription factor involved in multiple aspects of oncogenic proliferation that is used for evaluating prognosis and clinical grading of neuroblastoma. Furthermore, the addition of RA enhances all of these effects for all ECM stiffnesses tested. Together, our data strongly support the notion that

  4. α-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions

    PubMed Central

    Meacci, Giovanni; Wolfenson, Haguy; Liu, Shuaimin; Stachowiak, Matthew R.; Iskratsch, Thomas; Mathur, Anurag; Ghassemi, Saba; Gauthier, Nils; Tabdanov, Erdem; Lohner, James; Gondarenko, Alexander; Chander, Ashok C.; Roca-Cusachs, Pere; O’Shaughnessy, Ben; Hone, James; Sheetz, Michael P.

    2016-01-01

    During spreading and migration, the leading edges of cells undergo periodic protrusion–retraction cycles. The functional purpose of these cycles is unclear. Here, using submicrometer polydimethylsiloxane pillars as substrates for cell spreading, we show that periodic edge retractions coincide with peak forces produced by local contractile units (CUs) that assemble and disassemble along the cell edge to test matrix rigidity. We find that, whereas actin rearward flow produces a relatively constant force inward, the peak of local contractile forces by CUs scales with rigidity. The cytoskeletal protein α-actinin is shared between these two force-producing systems. It initially localizes to the CUs and subsequently moves inward with the actin flow. Knockdown of α-actinin causes aberrant rigidity sensing, loss of CUs, loss of protrusion–retraction cycles, and, surprisingly, enables the cells to proliferate on soft matrices. We present a model based on these results in which local CUs drive rigidity sensing and adhesion formation. PMID:27122603

  5. Extracellular matrix composition and rigidity regulate invasive behavior and response to PDT in 3D pancreatic tumor models

    NASA Astrophysics Data System (ADS)

    Cramer, Gwendolyn; El-Hamidi, Hamid; Jafari, Seyedehrojin; Jones, Dustin P.; Celli, Jonathan P.

    2016-03-01

    The composition and mechanical compliance of the extracellular matrix (ECM) have been shown to serve as regulators of tumor growth and invasive behavior. These effects may be particularly relevant in tumors of the pancreas, noted for a profound desmoplastic reaction and an abundance of stroma rich in ECM. In view of recent progress in the clinical implementation of photodynamic therapy (PDT) for pancreatic tumors, in this report we examine how ECM composition and rheological properties impact upon invasive behavior and response to PDT in 3D multicellular pancreatic tumor spheroids in ECM environments with characterized rheological properties. Tumor spheroids were cultured initially in attachment-free conditions to form millimeter-sized spheroids that were transplanted into reconstituted ECM microenvironments (Matrigel and Type I Collagen) that were characterized using bulk oscillatory shear rheology. Analysis of growth behavior shows that the soft collagen ECM promoted growth and extensive invasion and this microenvironment was used in subsequent assessment of PDT and chemotherapy response. Evaluation of treatment response revealed that primary tumor nodule growth is inhibited more effectively with PDT, while verteporfin PDT response is significantly enhanced in the ECM-infiltrating populations that are non-responsive to oxaliplatin chemotherapy. This finding is potentially significant, suggesting the potential for PDT to target these clinically problematic invasive populations that are associated with aggressive metastatic progression and chemoresistance. Experiments to further validate and identify the mechanistic basis of this observation are ongoing.

  6. Remodeling of aorta extracellular matrix as a result of transient high oxygen exposure in newborn rats: implication for arterial rigidity and hypertension risk.

    PubMed

    Huyard, Fanny; Yzydorczyk, Catherine; Castro, Michele M; Cloutier, Anik; Bertagnolli, Mariane; Sartelet, Hervé; Germain, Nathalie; Comte, Blandine; Schulz, Richard; DeBlois, Denis; Nuyt, Anne Monique

    2014-01-01

    Neonatal high-oxygen exposure leads to elevated blood pressure, microvascular rarefaction, vascular dysfunction and arterial (aorta) rigidity in adult rats. Whether structural changes are present in the matrix of aorta wall is unknown. Considering that elastin synthesis peaks in late fetal life in humans, and early postnatal life in rodents, we postulated that transient neonatal high-oxygen exposure can trigger premature vascular remodelling. Sprague Dawley rat pups were exposed from days 3 to 10 after birth to 80% oxygen (vs. room air control) and were studied at 4 weeks. Blood pressure and vasomotor response of the aorta to angiotensin II and to the acetylcholine analogue carbachol were not different between groups. Vascular superoxide anion production was similar between groups. There was no difference between groups in aortic cross sectional area, smooth muscle cell number or media/lumen ratio. In oxygen-exposed rats, aorta elastin/collagen content ratio was significantly decreased, the expression of elastinolytic cathepsin S was increased whereas collagenolytic cathepsin K was decreased. By immunofluorescence we observed an increase in MMP-2 and TIMP-1 staining in aortas of oxygen-exposed rats whereas TIMP-2 staining was reduced, indicating a shift in the balance towards degradation of the extra-cellular matrix and increased deposition of collagen. There was no significant difference in MMP-2 activity between groups as determined by gelatin zymography. Overall, these findings indicate that transient neonatal high oxygen exposure leads to vascular wall alterations (decreased elastin/collagen ratio and a shift in the balance towards increased deposition of collagen) which are associated with increased rigidity. Importantly, these changes are present prior to the elevation of blood pressure and vascular dysfunction in this model, and may therefore be contributory.

  7. Extracellular matrix structure.

    PubMed

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented.

  8. Making recombinant extracellular matrix proteins.

    PubMed

    Ruggiero, Florence; Koch, Manuel

    2008-05-01

    A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.

  9. Extracellular rigidity sensing by talin isoform–specific mechanical linkages

    PubMed Central

    Austen, Katharina; Ringer, Pia; Mehlich, Alexander; Chrostek-Grashoff, Anna; Kluger, Carleen; Klingner, Christoph; Sabass, Benedikt; Zent, Roy; Rief, Matthias; Grashoff, Carsten

    2015-01-01

    The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule–calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages upon cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton (pN) forces and bear, on average, 7–10 pN during cell adhesion depending on their association with f-actin and vinculin. Disruption of talin’s mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform-specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing. PMID:26523364

  10. Extracellular matrix and wound healing.

    PubMed

    Maquart, F X; Monboisse, J C

    2014-04-01

    Extracellular matrix has been known for a long time as an architectural support for the tissues. Many recent data, however, have shown that extracellular matrix macromolecules (collagens, elastin, glycosaminoglycans, proteoglycans and connective tissue glycoproteins) are able to regulate many important cell functions, such as proliferation, migration, protein synthesis or degradation, apoptosis, etc., making them able to play an important role in the wound repair process. Not only the intact macromolecules but some of their specific domains, that we called "Matrikines", are also able to regulate many cell activities. In this article, we will summarize main findings showing the effects of extracellular matrix macromolecules and matrikines on connective tissue and epithelial cells, particularly in skin, and their potential implication in the wound healing process. These examples show that extracellular matrix macromolecules or some of their specific domains may play a major role in wound healing. Better knowledge of these interactions may suggest new therapeutic targets in wound healing defects. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  11. Mechanotransduction and extracellular matrix homeostasis

    PubMed Central

    Humphrey, Jay D.; Dufresne, Eric R.; Schwartz, Martin A.

    2015-01-01

    Preface Soft connective tissues at steady state are yet dynamic; resident cells continually read environmental cues and respond to promote homeostasis, including maintenance of the mechanical properties of the extracellular matrix that are fundamental to cellular and tissue health. The mechanosensing process involves assessment of the mechanics of the matrix by the cells through integrins and the actomyosin cytoskeleton, and is followed by a mechano-regulation process that includes the deposition, rearrangement, or removal of matrix to maintain overall form and function. Progress toward understanding the molecular, cellular, and tissue scale effects that promote mechanical homeostasis has helped identify key questions for future research. PMID:25355505

  12. The evolution of extracellular matrix.

    PubMed

    Ozbek, Suat; Balasubramanian, Prakash G; Chiquet-Ehrismann, Ruth; Tucker, Richard P; Adams, Josephine C

    2010-12-01

    We present a perspective on the molecular evolution of the extracellular matrix (ECM) in metazoa that draws on research publications and data from sequenced genomes and expressed sequence tag libraries. ECM components do not function in isolation, and the biological ECM system or "adhesome" also depends on posttranslational processing enzymes, cell surface receptors, and extracellular proteases. We focus principally on the adhesome of internal tissues and discuss its origins at the dawn of the metazoa and the expansion of complexity that occurred in the chordate lineage. The analyses demonstrate very high conservation of a core adhesome that apparently evolved in a major wave of innovation in conjunction with the origin of metazoa. Integrin, CD36, and certain domains predate the metazoa, and some ECM-related proteins are identified in choanoflagellates as predicted sequences. Modern deuterostomes and vertebrates have many novelties and elaborations of ECM as a result of domain shuffling, domain innovations and gene family expansions. Knowledge of the evolution of metazoan ECM is important for understanding how it is built as a system, its roles in normal tissues and disease processes, and has relevance for tissue engineering, the development of artificial organs, and the goals of synthetic biology.

  13. The Evolution of Extracellular Matrix

    PubMed Central

    Özbek, Suat; Balasubramanian, Prakash G.; Chiquet-Ehrismann, Ruth; Tucker, Richard P.

    2010-01-01

    We present a perspective on the molecular evolution of the extracellular matrix (ECM) in metazoa that draws on research publications and data from sequenced genomes and expressed sequence tag libraries. ECM components do not function in isolation, and the biological ECM system or “adhesome” also depends on posttranslational processing enzymes, cell surface receptors, and extracellular proteases. We focus principally on the adhesome of internal tissues and discuss its origins at the dawn of the metazoa and the expansion of complexity that occurred in the chordate lineage. The analyses demonstrate very high conservation of a core adhesome that apparently evolved in a major wave of innovation in conjunction with the origin of metazoa. Integrin, CD36, and certain domains predate the metazoa, and some ECM-related proteins are identified in choanoflagellates as predicted sequences. Modern deuterostomes and vertebrates have many novelties and elaborations of ECM as a result of domain shuffling, domain innovations and gene family expansions. Knowledge of the evolution of metazoan ECM is important for understanding how it is built as a system, its roles in normal tissues and disease processes, and has relevance for tissue engineering, the development of artificial organs, and the goals of synthetic biology. PMID:21160071

  14. Extracellular Matrix: Functions in the Nervous System

    PubMed Central

    Barros, Claudia S.; Franco, Santos J.; Müller, Ulrich

    2011-01-01

    An astonishing number of extracellular matrix glycoproteins are expressed in dynamic patterns in the developing and adult nervous system. Neural stem cells, neurons, and glia express receptors that mediate interactions with specific extracellular matrix molecules. Functional studies in vitro and genetic studies in mice have provided evidence that the extracellular matrix affects virtually all aspects of nervous system development and function. Here we will summarize recent findings that have shed light on the specific functions of defined extracellular matrix molecules on such diverse processes as neural stem cell differentiation, neuronal migration, the formation of axonal tracts, and the maturation and function of synapses in the peripheral and central nervous system. PMID:21123393

  15. Extracellular Matrix and Liver Disease

    PubMed Central

    Arriazu, Elena; Ruiz de Galarreta, Marina; Cubero, Francisco Javier; Varela-Rey, Marta; Pérez de Obanos, María Pilar; Leung, Tung Ming; Lopategi, Aritz; Benedicto, Aitor; Abraham-Enachescu, Ioana

    2014-01-01

    Abstract Significance: The extracellular matrix (ECM) is a dynamic microenvironment that undergoes continuous remodeling, particularly during injury and wound healing. Chronic liver injury of many different etiologies such as viral hepatitis, alcohol abuse, drug-induced liver injury, obesity and insulin resistance, metabolic disorders, and autoimmune disease is characterized by excessive deposition of ECM proteins in response to persistent liver damage. Critical Issues: This review describes the main collagenous and noncollagenous components from the ECM that play a significant role in pathological matrix deposition during liver disease. We define how increased myofibroblasts (MF) from different origins are at the forefront of liver fibrosis and how liver cell-specific regulation of the complex scarring process occurs. Recent Advances: Particular attention is paid to the role of cytokines, growth factors, reactive oxygen species, and newly identified matricellular proteins in the regulation of fibrillar type I collagen, a field to which our laboratory has significantly contributed over the years. We compile data from recent literature on the potential mechanisms driving fibrosis resolution such as MF’ apoptosis, senescence, and reversal to quiescence. Future Directions: We conclude with a brief description of how epigenetics, an evolving field, can regulate the behavior of MF and of how new “omics” tools may advance our understanding of the mechanisms by which the fibrogenic response to liver injury occurs. Antioxid. Redox Signal. 21, 1078–1097. PMID:24219114

  16. Extracellular matrix proteins of dentine.

    PubMed

    Butler, W T; Ritchie, H H; Bronckers, A L

    1997-01-01

    Bone and dentine extracellular matrix proteins are similar, consisting primarily of type I collagen, acidic proteins and proteoglycans. Although collagen forms the lattice for deposition of calcium and phosphate for formation of carbonate apatite, the non-collagenous proteins are believed to control initiation and growth of the crystals. Despite this similarity, dentine contains three unique proteins apparently absent from bone and other tissue: dentine phosphophoryn (DPP), dentine matrix protein 1 (DMP1) and dentine sialoprotein (DSP). DPP and DMP1 are acidic phosphoproteins probably involved in the control of mineralization processes. DPP may localize in gap regions of collagen and initiate apatite crystal formation by binding large quantities of calcium in a conformation that promotes this process. Extensive studies have been conducted in our laboratory on the nature, biosynthesis, localization and gene structure of DSP. Immunolocalization studies showed that rat DSP, a 53 kDa sialic acid-rich glycoprotein, was synthesized by young and mature odontoblasts, and by dental pulp cells and pre-ameloblasts, but not by ameloblasts, osteoblasts, chondrocytes or other cell types. The cDNA sequence indicated that DSP was a 366-residue protein with several potential N-glycosylation sites, as well as phosphorylation sites, but that the amino acid sequence was dissimilar to that of other known proteins. Northern blot analysis detected several mRNA species near 4.6 and 1.5 kb, indicative of alternative splicing events. Evidence for two DSP genes was obtained, further complicating this picture. Recent in situ hybridization studies utilizing rat and mouse molars and incisors indicated that DSP mRNA was expressed by young odontoblasts and odontoblasts in animals of all ages. Transcripts were also observed in pre-ameloblasts. The expression of DSP mRNA ceased when these cells matured to become secretory ameloblasts. DSP transcripts were not detected in osteoblasts or other cell

  17. Cellular Traction Stresses Mediate Extracellular Matrix Degradation by Invadopodia

    PubMed Central

    Jerrell, Rachel J.; Parekh, Aron

    2014-01-01

    During tumorigenesis, matrix rigidity can drive oncogenic transformation via altered cellular proliferation and migration. Cells sense extracellular matrix (ECM) mechanical properties with intracellular tensile forces generated by actomyosin contractility. These contractile forces are transmitted to the matrix surface as traction stresses which mediate mechanical interactions with the ECM. Matrix rigidity has been shown to increase proteolytic ECM degradation by cytoskeletal structures known as invadopodia that are critical for cancer progression suggesting that cellular contractility promotes invasive behavior. However, both increases and decreases in traction stresses have been associated with metastatic behavior. Therefore, the role of cellular contractility in invasive migration leading to metastasis is unclear. To determine the relationship between cellular traction stresses and invadopodia activity, we characterized the invasive and contractile properties of an aggressive carcinoma cell line utilizing polyacrylamide gels of different rigidities. We found that ECM degradation and traction stresses were linear functions of matrix rigidity. Using calyculin A to augment myosin contractility, we also found that traction stresses were strongly predictive of ECM degradation. Overall, our data suggest that cellular force generation may play an important part in invasion and metastasis by mediating invadopodia activity in response to the mechanical properties of the tumor microenvironment. PMID:24412623

  18. Fragmentation of extracellular matrix by hypochlorous acid.

    PubMed Central

    Woods, Alan A; Davies, Michael J

    2003-01-01

    The interaction of extracellular matrix with cells regulates their adhesion, migration and proliferation, and it is believed that damage to vascular matrix components is a factor in the development of atherosclerosis. Evidence has been provided for a role for the haem enzyme MPO (myeloperoxidase), released by activated monocytes (and possibly macrophages), in oxidative events within the artery wall. As MPO is released extracellularly, and is highly basic, it might be expected to associate with poly-anionic matrix components thereby localizing damage to these materials. In this study the reaction of the MPO-derived oxidant hypochlorous acid (HOCl) with extracellular matrix from vascular smooth muscle cells and healthy pig arteries has been examined. HOCl is rapidly consumed by such matrix samples, with the formation of matrix-derived chloramines or chloramides. The yield of these intermediates increases with HOCl dose. These materials undergo a time- and temperature-dependent decay, which parallels the release of sugar and protein components from the treated matrix, consistent with these species being important intermediates. Matrix damage is enhanced by species that increase chloramine/chloramide decomposition, with copper and iron ions being effective catalysts, and decreased by compounds which scavenge chloramines/chloramides, or species derived from them. The effect of such matrix modifications on cellular behaviour is poorly understood, though it is known that changes in matrix materials can have profound effects on cell adhesion, proliferation, growth and phenotype. The observed matrix modifications reported here may therefore modulate cellular behaviour in diseases such as atherosclerosis where MPO-derived oxidants are generated. PMID:12911330

  19. MatrixDB, the extracellular matrix interaction database

    PubMed Central

    Chautard, Emilie; Fatoux-Ardore, Marie; Ballut, Lionel; Thierry-Mieg, Nicolas; Ricard-Blum, Sylvie

    2011-01-01

    MatrixDB (http://matrixdb.ibcp.fr) is a freely available database focused on interactions established by extracellular proteins and polysaccharides. Only few databases report protein–polysaccharide interactions and, to the best of our knowledge, there is no other database of extracellular interactions. MatrixDB takes into account the multimeric nature of several extracellular protein families for the curation of interactions, and reports interactions with individual polypeptide chains or with multimers, considered as permanent complexes, when appropriate. MatrixDB is a member of the International Molecular Exchange consortium (IMEx) and has adopted the PSI-MI standards for the curation and the exchange of interaction data. MatrixDB stores experimental data from our laboratory, data from literature curation, data imported from IMEx databases, and data from the Human Protein Reference Database. MatrixDB is focused on mammalian interactions, but aims to integrate interaction datasets of model organisms when available. MatrixDB provides direct links to databases recapitulating mutations in genes encoding extracellular proteins, to UniGene and to the Human Protein Atlas that shows expression and localization of proteins in a large variety of normal human tissues and cells. MatrixDB allows researchers to perform customized queries and to build tissue- and disease-specific interaction networks that can be visualized and analyzed with Cytoscape or Medusa. PMID:20852260

  20. MatrixDB, the extracellular matrix interaction database.

    PubMed

    Chautard, Emilie; Fatoux-Ardore, Marie; Ballut, Lionel; Thierry-Mieg, Nicolas; Ricard-Blum, Sylvie

    2011-01-01

    MatrixDB (http://matrixdb.ibcp.fr) is a freely available database focused on interactions established by extracellular proteins and polysaccharides. Only few databases report protein-polysaccharide interactions and, to the best of our knowledge, there is no other database of extracellular interactions. MatrixDB takes into account the multimeric nature of several extracellular protein families for the curation of interactions, and reports interactions with individual polypeptide chains or with multimers, considered as permanent complexes, when appropriate. MatrixDB is a member of the International Molecular Exchange consortium (IMEx) and has adopted the PSI-MI standards for the curation and the exchange of interaction data. MatrixDB stores experimental data from our laboratory, data from literature curation, data imported from IMEx databases, and data from the Human Protein Reference Database. MatrixDB is focused on mammalian interactions, but aims to integrate interaction datasets of model organisms when available. MatrixDB provides direct links to databases recapitulating mutations in genes encoding extracellular proteins, to UniGene and to the Human Protein Atlas that shows expression and localization of proteins in a large variety of normal human tissues and cells. MatrixDB allows researchers to perform customized queries and to build tissue- and disease-specific interaction networks that can be visualized and analyzed with Cytoscape or Medusa.

  1. Extracellular matrix signaling in morphogenesis and repair.

    PubMed

    Clause, Kelly C; Barker, Thomas H

    2013-10-01

    The extracellular matrix (ECM) is critically important for many cellular processes including growth, differentiation, survival, and morphogenesis. Cells remodel and reshape the ECM by degrading and reassembling it, playing an active role in sculpting their surrounding environment and directing their own phenotypes. Both mechanical and biochemical molecules influence ECM dynamics in multiple ways; by releasing small bioactive signaling molecules, releasing growth factors stored within the ECM, eliciting structural changes to matrix proteins which expose cryptic sites and by degrading matrix proteins directly. The dynamic reciprocal communication between cells and the ECM plays a fundamental roll in tissue development, homeostasis, and wound healing.

  2. Nanomechanics of the Cartilage Extracellular Matrix

    NASA Astrophysics Data System (ADS)

    Han, Lin; Grodzinsky, Alan J.; Ortiz, Christine

    2011-08-01

    Cartilage is a hydrated biomacromolecular fiber composite located at the ends of long bones that enables proper joint lubrication, articulation, loading, and energy dissipation. Degradation of extracellular matrix molecular components and changes in their nanoscale structure greatly influence the macroscale behavior of the tissue and result in dysfunction with age, injury, and diseases such as osteoarthritis. Here, the application of the field of nanomechanics to cartilage is reviewed. Nanomechanics involves the measurement and prediction of nanoscale forces and displacements, intra- and intermolecular interactions, spatially varying mechanical properties, and other mechanical phenomena existing at small length scales. Experimental nanomechanics and theoretical nanomechanics have been applied to cartilage at varying levels of material complexity, e.g., nanoscale properties of intact tissue, the matrix associated with single cells, biomimetic molecular assemblies, and individual extracellular matrix biomolecules (such as aggrecan, collagen, and hyaluronan). These studies have contributed to establishing a fundamental mechanism-based understanding of native and engineered cartilage tissue function, quality, and pathology.

  3. Cell stiffness, contractile stress and the role of extracellular matrix

    SciTech Connect

    An, Steven S.; Kim, Jina; Ahn, Kwangmi; Trepat, Xavier; Drake, Kenneth J.; Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne; Fredberg, Jeffrey J.; Biswal, Shyam

    2009-05-15

    Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

  4. Perturbation of red blood cell membrane rigidity by extracellular ligands.

    PubMed

    Paulitschke, M; Nash, G B; Anstee, D J; Tanner, M J; Gratzer, W B

    1995-07-01

    It is known that binding of extracellular antibodies against the major sialoglycoprotein, glycophorin A, reduced the deformability of the red blood cell membrane. This has been taken to result from new or altered interactions between the glycophorin A and the membrane skeleton. We have shown by means of the micropipette aspiration technique that antibodies against the preponderant transmembrane protein, band 3, induce similar effects. A definite but much smaller reduction in elasticity of the membrane is engendered by univalent Fab fragments of the anti-band 3 antibodies. By examining cells genetically devoid of glycophorin A or containing a variant of this constituent, truncated at the inner membrane surface, we have shown that the anti-band 3 antibodies do not act through the band 3-associated glycophorin A. We examined the effect of anti-glycophorin A antibodies on homozygous Wr(a+b-) cells, in which an amino acid replacement in band 3 annihilates the Wright b (Wrb) epitope (comprising sequence elements of glycophorin A and band 3) and thus, by implication disrupts or perturbs the band 3-glycophorin A interaction; these cells show a much smaller response to an anti-glycophorin A antibody than do normal controls. We infer that in this case anti-glycophorin A antibodies exert their rigidifying effect through the associated band 3. Another anti-glycophorin A antibody, directed against an epitope remote from the membrane surface, however, increases the rigidity of both Wr(a+b-) and normal cells. This implies that not all antibodies act in the same manner in modifying the membrane mechanical properties. The effect exerted by anti-band 3 antibodies appears not to be transmitted through the band 3-ankyrin-spectrin pathway because the rigidifying effect of the intact antibody persists at alkaline pH, at which there is evidence that the ankyrin-band 3 link is largely dissociated. The large difference between the effects of saturating concentrations of the divalent and

  5. Involvement of extracellular matrix constituents in breast cancer

    SciTech Connect

    Lochter, Andre; Bissell, Mina J

    1995-06-01

    It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

  6. Airway and Extracellular Matrix Mechanics in COPD

    PubMed Central

    Bidan, Cécile M.; Veldsink, Annemiek C.; Meurs, Herman; Gosens, Reinoud

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases worldwide, and is characterized by airflow obstruction that is not fully reversible with treatment. Even though airflow obstruction is caused by airway smooth muscle contraction, the extent of airway narrowing depends on a range of other structural and functional determinants that impact on active and passive tissue mechanics. Cells and extracellular matrix in the airway and parenchymal compartments respond both passively and actively to the mechanical stimulation induced by smooth muscle contraction. In this review, we summarize the factors that regulate airway narrowing and provide insight into the relative contributions of different constituents of the extracellular matrix and their biomechanical impact on airway obstruction. We then review the changes in extracellular matrix composition in the airway and parenchymal compartments at different stages of COPD, and finally discuss how these changes impact airway narrowing and the development of airway hyperresponsiveness. Finally, we position these data in the context of therapeutic research focused on defective tissue repair. As a conclusion, we propose that future works should primarily target mild or early COPD, prior to the widespread structural changes in the alveolar compartment that are more characteristic of severe COPD. PMID:26696894

  7. Pneumococcal MSCRAMM targeting of the extracellular matrix

    PubMed Central

    Paterson, Gavin K.; Orihuela, Carlos J.

    2010-01-01

    The attachment of bacteria to host cells and tissues and their subsequent invasion and dissemination are key processes during disease pathogenesis. In this issue of Molecular Microbiology, Jensch and co-workers provide further molecular insight into these events during infection with the Gram-positive bacterium Streptococcus pneumoniae. Their characterization of PavB, a bacterial surface protein with orthologues in other streptococci, shows it to bind the extracellar matrix components fibronection and plasminogen by virtue of repetitive sequences designated Streptococcal Surface Repeats (SSURE). In mice, a pavB mutant showed reduced nasopharyngeal colonisation and was attenuated in a lung infection model. As discussed here in the context of the pneumococcus, the study of PavB highlights the central role during microbal pathogenesis of targetting the extracellular matrix by so-called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). PMID:20444102

  8. Achondrogenesis type II, abnormalities of extracellular matrix.

    PubMed

    Horton, W A; Machado, M A; Chou, J W; Campbell, D

    1987-09-01

    Immune and lectin histochemical and microchemical methods were employed to study growth cartilage from seven cases of achondrogenesis type II (Langer-Saldino). The normal architecture of the epiphyseal and growth plate cartilage was replaced by a morphologically heterogeneous tissue. Some areas were comprised of vascular canals surrounded by extensive fibrous tissue and enlarged cells that had the appearance and histochemical characteristics of hypertrophic chondrocytes. Other areas contained a mixture of cells ranging from small to the enlarged chondrocytes. The extracellular matrix in the latter areas was more abundant and had characteristics of both precartilage mesenchymal matrix and typical cartilage matrix; it contained types I and II collagen, cartilage proteoglycan, fibronectin, and peanut agglutinin binding glycoconjugate(s). Peptide mapping of cyanogen bromide cartilage collagen peptides revealed the presence of types I and II collagen. These observations could be explained by a defect in the biosynthesis of type II collagen or in chondrocyte differentiation.

  9. Bioengineering Human Myocardium on Native Extracellular Matrix

    PubMed Central

    Guyette, Jacques P.; Charest, Jonathan M; Mills, Robert W; Jank, Bernhard J.; Moser, Philipp T.; Gilpin, Sarah E.; Gershlak, Joshua R.; Okamoto, Tatsuya; Gonzalez, Gabriel; Milan, David J.; Gaudette, Glenn R.; Ott, Harald C.

    2015-01-01

    Rationale More than 25 million individuals suffer from heart failure worldwide, with nearly 4,000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only about 2,500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. Objective The objective of this study is to translate previous work to human scale and clinically relevant cells, for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human iPS-derived cardiac myocytes. Methods and Results To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiac myocytes derived from non-transgenic human induced pluripotent stem cells (iPSCs) and generated tissues of increasing three-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole heart scaffolds with human iPSC-derived cardiac myocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue, showed electrical conductivity, left ventricular pressure development, and metabolic function. Conclusions Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human iPS-derived cardiac myocytes, and enable

  10. The evolution of metazoan extracellular matrix

    PubMed Central

    2012-01-01

    The modular domain structure of extracellular matrix (ECM) proteins and their genes has allowed extensive exon/domain shuffling during evolution to generate hundreds of ECM proteins. Many of these arose early during metazoan evolution and have been highly conserved ever since. Others have undergone duplication and divergence during evolution, and novel combinations of domains have evolved to generate new ECM proteins, particularly in the vertebrate lineage. The recent sequencing of several genomes has revealed many details of this conservation and evolution of ECM proteins to serve diverse functions in metazoa. PMID:22431747

  11. Extracellular matrix alterations in the Peyronie's disease.

    PubMed

    Watanabe, Marcelo Silva; Theodoro, Thérèse Rachel; Coelho, Natália Lima; Mendes, Aline; Leonel, Monica Luzia Pereira; Mader, Ana Maria; Nader, Helena Bonciani; Glina, Sidney; Pinhal, Maria Aparecida Silva

    2017-07-01

    Peyronie's disease is characterized by fibrous plaque formation of the tunica albuginea, causing penile deformity and fertility problems. The aim of the present study was to investigate alterations in the extracellular matrix in Peyronie's disease. The study used tissues collected by surgical procedure from individuals that presented a well-established disease, while control samples were obtained by biopsies of fresh cadavers. Immunohistochemistry analysis followed by digital quantification was performed to evaluate TGF-β, heparanases and metalloproteinases (MMPs). The profile of sulfated glycosaminoglycans, chondroitin sulfate and dermatan sulfate was determined by agarose gel electrophoresis, while hyaluronic acid quantification was obtained by an ELISA-like assay. The expression of mRNA was investigated for syndecan-1 proteoglycan (Syn-1), interleukine-6 (IL-6), hyaluronic acid synthases, and hyaluronidases. Pathologic features showed decreased apoptosis and blood vessel number in Peyronie's tissues. TGF-β and IL-6 were significantly enhanced in Peyronie's disease. There was an increased expression of heparanases, though no alteration was observed for MMPs. Hyaluronic acid as well as hyaluronic acid synthases, hyaluronidases, and dermatan sulfate were not changed, while the level of chondroitin sulfate was significantly (P = 0.008, Mann-Whitney test) increased in Peyronie's samples. Heparanases and sulfated glycosaminoglycans seem to be involved in extracellular matrix alterations in Peyronie's disease.

  12. Decellularized musculofascial extracellular matrix for tissue engineering

    PubMed Central

    Wang, Lina; Johnson, Joshua A; Chang, David W.; Zhang, Qixu

    2016-01-01

    Ideal scaffolds that represent native extracellular matrix (ECM) properties of musculofascial tissues have great importance in musculofascial tissue engineering. However, detailed characterization of musculofascial tissues’ ECM (particularly, of fascia) from large animals is still lacking. In this study, we developed a decellularization protocol for processing pig composite musculofascial tissues. Decellularized muscle (D-muscle) and decellularized fascia (D-fascia), which are two important components of decellularized musculofascial extracellular matrix (DMM), were comprehensively characterized. D-muscle and D-fascia retained intact three-dimensional architecture, strong mechanical properties, and bioactivity of compositions such as collagen, laminin, glycosaminoglycan, and vascular endothelial growth factor. D-muscle and D-fascia provided a compatible niche for human adipose-derived stem cell integration and proliferation. Heterotopic and orthotopic implantation of D-muscle and D-fascia in a rodent model further proved their biocompatibility and myogenic properties during the remodeling process. The differing characteristics of D-muscle from D-fascia (e.g., D-muscle’s strong pro-angiogenic and pro-myogenic properties vs. D-fascia’s strong mechanical properties) indicate different clinical application opportunities of D-muscle vs. D-fascia scaffolds. DMM comprising muscle and fascia ECM as a whole unit can thus provide not only a clinically translatable platform for musculofascial tissue repair and regeneration but also a useful standard for scaffold design in musculofascial tissue engineering. PMID:23347834

  13. Extracellular Matrix, a Hard Player in Angiogenesis

    PubMed Central

    Mongiat, Maurizio; Andreuzzi, Eva; Tarticchio, Giulia; Paulitti, Alice

    2016-01-01

    The extracellular matrix (ECM) is a complex network of proteins, glycoproteins, proteoglycans, and polysaccharides. Through multiple interactions with each other and the cell surface receptors, not only the ECM determines the physical and mechanical properties of the tissues, but also profoundly influences cell behavior and many physiological and pathological processes. One of the functions that have been extensively explored is its impingement on angiogenesis. The strong impact of the ECM in this context is both direct and indirect by virtue of its ability to interact and/or store several growth factors and cytokines. The aim of this review is to provide some examples of the complex molecular mechanisms that are elicited by these molecules in promoting or weakening the angiogenic processes. The scenario is intricate, since matrix remodeling often generates fragments displaying opposite effects compared to those exerted by the whole molecules. Thus, the balance will tilt towards angiogenesis or angiostasis depending on the relative expression of pro- or anti-angiogenetic molecules/fragments composing the matrix of a given tissue. One of the vital aspects of this field of research is that, for its endogenous nature, the ECM can be viewed as a reservoir to draw from for the development of new more efficacious therapies to treat angiogenesis-dependent pathologies. PMID:27809279

  14. The extracellular matrix in breast cancer.

    PubMed

    Insua-Rodríguez, Jacob; Oskarsson, Thordur

    2016-02-01

    The extracellular matrix (ECM) is increasingly recognized as an important regulator in breast cancer. ECM in breast cancer development features numerous changes in composition and organization when compared to the mammary gland under homeostasis. Matrix proteins that are induced in breast cancer include fibrillar collagens, fibronectin, specific laminins and proteoglycans as well as matricellular proteins. Growing evidence suggests that many of these induced ECM proteins play a major functional role in breast cancer progression and metastasis. A number of the induced ECM proteins have moreover been shown to be essential components of metastatic niches, promoting stem/progenitor signaling pathways and metastatic growth. ECM remodeling enzymes are also markedly increased, leading to major changes in the matrix structure and biomechanical properties. Importantly, several ECM components and ECM remodeling enzymes are specifically induced in breast cancer or during tissue regeneration while healthy tissues under homeostasis express exceedingly low levels. This may indicate that ECM and ECM-associated functions may represent promising drug targets against breast cancer, providing important specificity that could be utilized when developing therapies. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Extracellular matrix motion and early morphogenesis.

    PubMed

    Loganathan, Rajprasad; Rongish, Brenda J; Smith, Christopher M; Filla, Michael B; Czirok, Andras; Bénazéraf, Bertrand; Little, Charles D

    2016-06-15

    For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale 'total' cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis. © 2016. Published by The Company of Biologists Ltd.

  16. Extracellular Matrix Revisited: Roles in Tissue Engineering

    PubMed Central

    2016-01-01

    The extracellular matrix (ECM) is a heterogeneous, connective network composed of fibrous glycoproteins that coordinate in vivo to provide the physical scaffolding, mechanical stability, and biochemical cues necessary for tissue morphogenesis and homeostasis. This review highlights some of the recently raised aspects of the roles of the ECM as related to the fields of biophysics and biomedical engineering. Fundamental aspects of focus include the role of the ECM as a basic cellular structure, for novel spontaneous network formation, as an ideal scaffold in tissue engineering, and its essential contribution to cell sheet technology. As these technologies move from the laboratory to clinical practice, they are bound to shape the vast field of tissue engineering for medical transplantations. PMID:27230457

  17. Why regenerative medicine needs an extracellular matrix.

    PubMed

    Prestwich, Glenn D; Healy, Kevin E

    2015-01-01

    Regenerative medicine is now coming of age. Many attempts at cell therapy have failed to show significant efficacy, and the umbrella term 'stem cell therapy' is perceived in some quarters as hype or just expensive and unnecessary medical tourism. Here we present a short editorial in three parts. First, we examine the importance of using a semisynthetic extracellular matrix (ECM) mimetic, or sECM, to deliver and retain therapeutic cells at the site of administration. Second, we describe one approach in which biophysical and biochemical properties are tailored to each tissue type, which we call "design for optimal functionality." Third, we describe an alternative approach to sECM design and implementation, called "design for simplicity," in which a deconstructed, minimalist sECM is employed and biology is allowed to perform the customization in situ. We opine that an sECM, whether minimal or instructive, is an essential contributor to improve the outcomes of cell-based therapies.

  18. Defining the extracellular matrix using proteomics

    PubMed Central

    Byron, Adam; Humphries, Jonathan D; Humphries, Martin J

    2013-01-01

    The cell microenvironment has a profound influence on the behaviour, growth and survival of cells. The extracellular matrix (ECM) provides not only mechanical and structural support to cells and tissues but also binds soluble ligands and transmembrane receptors to provide spatial coordination of signalling processes. The ability of cells to sense the chemical, mechanical and topographical features of the ECM enables them to integrate complex, multiparametric information into a coherent response to the surrounding microenvironment. Consequently, dysregulation or mutation of ECM components results in a broad range of pathological conditions. Characterization of the composition of ECM derived from various cells has begun to reveal insights into ECM structure and function, and mechanisms of disease. Proteomic methodologies permit the global analysis of subcellular systems, but extracellular and transmembrane proteins present analytical difficulties to proteomic strategies owing to the particular biochemical properties of these molecules. Here, we review advances in proteomic approaches that have been applied to furthering our understanding of the ECM microenvironment. We survey recent studies that have addressed challenges in the analysis of ECM and discuss major outcomes in the context of health and disease. In addition, we summarize efforts to progress towards a systems-level understanding of ECM biology. PMID:23419153

  19. Crosstalk between glia, extracellular matrix and neurons.

    PubMed

    Song, Inseon; Dityatev, Alexander

    2017-03-08

    Extracellular matrix (ECM) molecules in the central nervous system form highly organized ECM structures around cell somata, axon initial segments, and synapses and play prominent roles in early development by guiding cell migration, neurite outgrowth and synaptogenesis, and by regulating closure of the critical period of development, synaptic plasticity and stability, cognitive flexibility, and axonal regeneration in adults. Major components of neural ECM, including chondroitin sulfate proteoglycans (CSPGs), tenascin-R and hyaluronic acid, are synthesized by both neurons and glial cells. The expression of these molecules is dynamically regulated during brain development in physiological conditions, shaping both neuronal and glial functions through multitude of molecular mechanisms. Upregulation of particular CSPGs and other ECM molecules, in particular by reactive astrocytes, after CNS injuries, during aging, neuroinflammation, and neurodegeneration on the one hand results in formation of growth-impermissive environment and impaired synaptic plasticity. On the other hand, ECM appeared to have a neuroprotective effect, at least in the form of perineuronal nets. CSPGs-degrading matrix metalloproteinases (MMPs) and several members of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family of proteases are secreted by neurons and glia and may drive neural ECM remodeling in physiological conditions as well as after brain injury and other brain disorders. Thus, targeting expression of specific ECM molecules, associated glycans and degrading enzymes may lead to development of new therapeutic strategies promoting regeneration and synaptic plasticity.

  20. Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

    PubMed Central

    Zucker, Stanley; Hymowitz, Michelle; Rollo, Ellen E.; Mann, Richard; Conner, Cathleen E.; Cao, Jian; Foda, Hussein D.; Tompkins, David C.; Toole, Bryan P.

    2001-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs. PMID:11395366

  1. Molecular adhesion between cartilage extracellular matrix macromolecules.

    PubMed

    Rojas, Fredrick P; Batista, Michael A; Lindburg, C Alexander; Dean, Delphine; Grodzinsky, Alan J; Ortiz, Christine; Han, Lin

    2014-03-10

    In this study, we investigated the molecular adhesion between the major constituents of cartilage extracellular matrix, namely, the highly negatively charged proteoglycan aggrecan and the type II/IX/XI fibrillar collagen network, in simulated physiological conditions. Colloidal force spectroscopy was applied to measure the maximum adhesion force and total adhesion energy between aggrecan end-attached spherical tips (end radius R ≈ 2.5 μm) and trypsin-treated cartilage disks with undamaged collagen networks. Studies were carried out in various aqueous solutions to reveal the physical factors that govern aggrecan-collagen adhesion. Increasing both ionic strength and [Ca(2+)] significantly increased adhesion, highlighting the importance of electrostatic repulsion and Ca(2+)-mediated ion bridging effects. In addition, we probed how partial enzymatic degradation of the collagen network, which simulates osteoarthritic conditions, affects the aggrecan-collagen interactions. Interestingly, we found a significant increase in aggrecan-collagen adhesion even when there were no detectable changes at the macro- or microscales. It is hypothesized that the aggrecan-collagen adhesion, together with aggrecan-aggrecan self-adhesion, works synergistically to determine the local molecular deformability and energy dissipation of the cartilage matrix, in turn, affecting its macroscopic tissue properties.

  2. Extracellular Matrix Biomarkers for Diagnosis, Prognosis, Imaging, and Targeting

    DTIC Science & Technology

    2015-09-01

    use in diagnosis, prognosis, early detection, in situ imaging and, eventually, targeting of breast cancer metastases, which are the major cause of...the management and treatment of metastatic breast cancer . 15. SUBJECT TERMS Breast Cancer , Metastasis, Extracellular Matrix, Tumor Microenvironment...KEYWORDS: Breast Cancer , Metastasis, Extracellular Matrix, Tumor Microenvironment, Tumor Heterogeneity 3. ACCOMPLISHMENTS: HYNES lab: Major Task 1

  3. Extracellular matrix in obesity - cancer interactions.

    PubMed

    Barreto, Stephany C; Hopkins, Christina A; Bhowmick, Meghnad; Ray, Amitabha

    2015-05-01

    Obesity or overweight is a risk factor for several health disorders such as type 2 diabetes, hypertension, and certain cancers. Furthermore, obesity affects almost all body systems including the extracellular matrix (ECM) by generating a pro-inflammatory environment, which are associated with abnormal secretions of several cytokines or hormonal substances, for example, insulin-like growth factors (IGFs), leptin, and sex hormones. These chemical mediators most likely have a great impact on the ECM. Accumulating evidence suggests that both obesity and ECM can influence tumor growth and progression through a number of chemical mediators. Conversely, cells in the connective tissue, namely fibroblasts and macrophages, support and aggravate the inflammatory situation in obesity by releasing several cytokines or growth factors such as vascular endothelial growth factor, epidermal growth factor, and transforming growth factor-beta (TGF-β). A wide range of functions are performed by TGF-β in normal health and pathological conditions including tumorigenesis. Breast cancer in postmenopausal women is a classic example of obesity-related cancer wherein several of these conditions, for example, higher levels of pro-inflammatory cytokines, impairment in the regulation of estrogen and growth factors, and dysregulation of different ECM components may favor the neoplastic process. Aberrant expressions of ECM components such as matrix metalloproteinases or matricellular proteins in both obesity and cancer have been reported by many studies. Nonstructural matricellular proteins, viz., thrombospondins, secreted protein acidic and rich in cysteine (SPARC), and Cyr61-CTGF-Nov (CCN), which function as modulators of cell-ECM interactions, exhibit protean behavior in cancer. Precise understanding of ECM biology can provide potential therapeutic targets to combat obesity-related pathologies.

  4. Vascular Extracellular Matrix and Arterial Mechanics

    PubMed Central

    WAGENSEIL, JESSICA E.; MECHAM, ROBERT P.

    2009-01-01

    An important factor in the transition from an open to a closed circulatory system was a change in vessel wall structure and composition that enabled the large arteries to store and release energy during the cardiac cycle. The component of the arterial wall in vertebrates that accounts for these properties is the elastic fiber network organized by medial smooth muscle. Beginning with the onset of pulsatile blood flow in the developing aorta, smooth muscle cells in the vessel wall produce a complex extracellular matrix (ECM) that will ultimately define the mechanical properties that are critical for proper function of the adult vascular system. This review discusses the structural ECM proteins in the vertebrate aortic wall and will explore how the choice of ECM components has changed through evolution as the cardiovascular system became more advanced and pulse pressure increased. By correlating vessel mechanics with physiological blood pressure across animal species and in mice with altered vessel compliance, we show that cardiac and vascular development are physiologically coupled, and we provide evidence for a universal elastic modulus that controls the parameters of ECM deposition in vessel wall development. We also discuss mechanical models that can be used to design better tissue-engineered vessels and to test the efficacy of clinical treatments. PMID:19584318

  5. Micro- and macrorheology of jellyfish extracellular matrix.

    PubMed

    Gambini, Camille; Abou, Bérengère; Ponton, Alain; Cornelissen, Annemiek J M

    2012-01-04

    Mechanical properties of the extracellular matrix (ECM) play a key role in tissue organization and morphogenesis. Rheological properties of jellyfish ECM (mesoglea) were measured in vivo at the cellular scale by passive microrheology techniques: microbeads were injected in jellyfish ECM and their Brownian motion was recorded to determine the mechanical properties of the surrounding medium. Microrheology results were compared with macrorheological measurements performed with a shear rheometer on slices of jellyfish mesoglea. We found that the ECM behaved as a viscoelastic gel at the macroscopic scale and as a much softer and heterogeneous viscoelastic structure at the microscopic scale. The fibrous architecture of the mesoglea, as observed by differential interference contrast and scanning electron microscopy, was in accord with these scale-dependent mechanical properties. Furthermore, the evolution of the mechanical properties of the ECM during aging was investigated by measuring microrheological properties at different jellyfish sizes. We measured that the ECM in adult jellyfish was locally stiffer than in juvenile ones. We argue that this stiffening is a consequence of local aggregations of fibers occurring gradually during aging of the jellyfish mesoglea and is enhanced by repetitive muscular contractions of the jellyfish.

  6. Engineering hydrogels as extracellular matrix mimics

    PubMed Central

    Geckil, Hikmet; Xu, Feng; Zhang, Xiaohui; Moon, SangJun

    2010-01-01

    Extracellular matrix (ECM) is a complex cellular environment consisting of proteins, proteoglycans, and other soluble molecules. ECM provides structural support to mammalian cells and a regulatory milieu with a variety of important cell functions, including assembling cells into various tissues and organs, regulating growth and cell–cell communication. Developing a tailored in vitro cell culture environment that mimics the intricate and organized nanoscale meshwork of native ECM is desirable. Recent studies have shown the potential of hydrogels to mimic native ECM. Such an engineered native-like ECM is more likely to provide cells with rational cues for diagnostic and therapeutic studies. The research for novel biomaterials has led to an extension of the scope and techniques used to fabricate biomimetic hydrogel scaffolds for tissue engineering and regenerative medicine applications. In this article, we detail the progress of the current state-of-the-art engineering methods to create cell-encapsulating hydrogel tissue constructs as well as their applications in in vitro models in biomedicine. PMID:20394538

  7. Extracellular matrix components in peripheral nerve regeneration.

    PubMed

    Gonzalez-Perez, Francisco; Udina, Esther; Navarro, Xavier

    2013-01-01

    Injured axons of the peripheral nerve are able to regenerate and, eventually, reinnervate target organs. However, functional recovery is usually poor after severe nerve injuries. The switch of Schwann cells to a proliferative state, secretion of trophic factors, and the presence of extracellular matrix (ECM) molecules (such as collagen, laminin, or fibronectin) in the distal stump are key elements to create a permissive environment for axons to grow. In this review, we focus attention on the ECM components and their tropic role in axonal regeneration. These components can also be used as molecular cues to guide the axons through artificial nerve guides in attempts to better mimic the natural environment found in a degenerating nerve. Most used scaffolds tested are based on natural molecules that form the ECM, but use of synthetic polymers and functionalization of hydrogels are bringing new options. Progress in tissue engineering will eventually lead to the design of composite artificial nerve grafts that may replace the use of autologous nerve grafts to sustain regeneration over long gaps.

  8. Extracellular matrix, supramolecular organisation and shape.

    PubMed Central

    Scott, J E

    1995-01-01

    Connective tissue function is defined as the formation and maintenance of shape, without which centralised physiologies (circulatory, digestive or nervous) could not have evolved. Two elements, inextensible (collagenous) fibrils and compression-resistant interfibrillar soluble polymers (proteoglycans), cope with all usual stresses. Relationships between the two are highly specific, as demonstrated by electron histochemistry based on Cupromeronic blue and critical electrolyte concentration (CEC) methodologies. Recent ideas on (1) the protofibrillar or modular structure of collagen fibrils, (2) the nature of specific binding sites for proteoglycans on fibrils, and (3) fundamental similarities in secondary and tertiary structures of the glycosaminoglycans (hyaluronan, chondroitin, keratan and dermatan sulphates) are described. They have greatly illuminated the study of extracellular matrix structure and function in normal, pathological (osteogenesis imperfecta) and ageing tissues. The small proteoglycans are proposed to be tissue organisers, orienting and ordering the collagen fibrils--thus shaping the tissue at a molecular and ultimately macro level. These interfibrillar structures are based on their bifunctional character, the protein parts binding to collagen fibrils at specific sites and the glycosaminoglycans duplexing and aggregating to hold the proteins and hence the collagen fibrils at defined distances from each other, rather like yardsticks. Examples of the way these functions work in specific tissues are drawn from the cornea and vitreous humour of the eye and developing tendon. Images Fig. 3 (cont.) Fig. 3 PMID:7591990

  9. Micro- and Macrorheology of Jellyfish Extracellular Matrix

    PubMed Central

    Gambini, Camille; Abou, Bérengère; Ponton, Alain; Cornelissen, Annemiek J.M.

    2012-01-01

    Mechanical properties of the extracellular matrix (ECM) play a key role in tissue organization and morphogenesis. Rheological properties of jellyfish ECM (mesoglea) were measured in vivo at the cellular scale by passive microrheology techniques: microbeads were injected in jellyfish ECM and their Brownian motion was recorded to determine the mechanical properties of the surrounding medium. Microrheology results were compared with macrorheological measurements performed with a shear rheometer on slices of jellyfish mesoglea. We found that the ECM behaved as a viscoelastic gel at the macroscopic scale and as a much softer and heterogeneous viscoelastic structure at the microscopic scale. The fibrous architecture of the mesoglea, as observed by differential interference contrast and scanning electron microscopy, was in accord with these scale-dependent mechanical properties. Furthermore, the evolution of the mechanical properties of the ECM during aging was investigated by measuring microrheological properties at different jellyfish sizes. We measured that the ECM in adult jellyfish was locally stiffer than in juvenile ones. We argue that this stiffening is a consequence of local aggregations of fibers occurring gradually during aging of the jellyfish mesoglea and is enhanced by repetitive muscular contractions of the jellyfish. PMID:22225792

  10. Extracellular matrix mechanics in lung parenchymal diseases.

    PubMed

    Suki, Béla; Bates, Jason H T

    2008-11-30

    In this review, we examine how the extracellular matrix (ECM) of the lung contributes to the overall mechanical properties of the parenchyma, and how these properties change in disease. The connective tissues of the lung are composed of cells and ECM, which includes a variety of biological macromolecules and water. The macromolecules that are most important in determining the mechanical properties of the ECM are collagen, elastin, and proteoglycans. We first discuss the various components of the ECM and how their architectural organization gives rise to the mechanical properties of the parenchyma. Next, we examine how mechanical forces can affect the physiological functioning of the lung parenchyma. Collagen plays an especially important role in determining the homeostasis and cellular responses to injury because it is the most important load-bearing component of the parenchyma. We then demonstrate how the concept of percolation can be used to link microscopic pathologic alterations in the parenchyma to clinically measurable lung function during the progression of emphysema and fibrosis. Finally, we speculate about the possibility of using targeted tissue engineering to optimize treatment of these two major lung diseases.

  11. Magnetic spectroscopy of nanoparticle Brownian motion measurement of microenvironment matrix rigidity.

    PubMed

    Weaver, John B; Rauwerdink, Kristen M; Rauwerdink, Adam M; Perreard, Irina M

    2013-12-01

    The rigidity of the extracellular matrix and of the integrin links to the cytoskeleton regulates signaling cascades, controlling critical aspects of cancer progression including metastasis and angiogenesis. We demonstrate that the matrix stiffness can be monitored using magnetic spectroscopy of nanoparticle Brownian motion (MSB). We measured the MSB signal from nanoparticles bound to large dextran polymers. The number of glutaraldehyde induced cross-links was used as a surrogate for material stiffness. There was a highly statistically significant change in the MSB signal with the number of cross-links especially prominent at higher frequencies. The p-values were all highly significant. We conclude that the MSB signal can be used to identify and monitor changes in the stiffness of the local matrix to which the nanoparticles are bound.

  12. The extracellular matrix of plants: Molecular, cellular and developmental biology

    SciTech Connect

    1996-12-31

    A symposium entitled ``The Extracellular Matrix of Plants: Molecular, Cellular and Developmental Biology was held in Tamarron, Colorado, March 15--21, 1996. The following topics were explored in addresses by 43 speakers: structure and biochemistry of cell walls; biochemistry, molecular biology and biosynthesis of lignin; secretory pathway and synthesis of glycoproteins; biosynthesis of matrix polysaccharides, callose and cellulose; role of the extracellular matrix in plant growth and development; plant cell walls in symbiosis and pathogenesis.

  13. Lung extracellular matrix and redox regulation.

    PubMed

    Watson, Walter H; Ritzenthaler, Jeffrey D; Roman, Jesse

    2016-08-01

    Pulmonary fibrosis affects millions worldwide and, even though there has been a significant investment in understanding the processes involved in wound healing and maladaptive repair, a complete understanding of the mechanisms responsible for lung fibrogenesis eludes us, and interventions capable of reversing or halting disease progression are not available. Pulmonary fibrosis is characterized by the excessive expression and uncontrolled deposition of extracellular matrix (ECM) proteins resulting in erosion of the tissue structure. Initially considered an 'end-stage' process elicited after injury, these events are now considered pathogenic and are believed to contribute to the course of the disease. By interacting with integrins capable of signal transduction and by influencing tissue mechanics, ECM proteins modulate processes ranging from cell adhesion and migration to differentiation and growth factor expression. In doing so, ECM proteins help orchestrate complex developmental processes and maintain tissue homeostasis. However, poorly controlled deposition of ECM proteins promotes inflammation, fibroproliferation, and aberrant differentiation of cells, and has been implicated in the pathogenesis of pulmonary fibrosis, atherosclerosis and cancer. Considering their vital functions, ECM proteins are the target of investigation, and oxidation-reduction (redox) reactions have emerged as important regulators of the ECM. Oxidative stress invariably accompanies lung disease and promotes ECM expression directly or through the overproduction of pro-fibrotic growth factors, while affecting integrin binding and activation. In vitro and in vivo investigations point to redox reactions as targets for intervention in pulmonary fibrosis and related disorders, but studies in humans have been disappointing probably due to the narrow impact of the interventions tested, and our poor understanding of the factors that regulate these complex reactions. This review is not meant to

  14. Lung extracellular matrix and redox regulation

    PubMed Central

    Watson, Walter H.; Ritzenthaler, Jeffrey D.; Roman, Jesse

    2016-01-01

    Pulmonary fibrosis affects millions worldwide and, even though there has been a significant investment in understanding the processes involved in wound healing and maladaptive repair, a complete understanding of the mechanisms responsible for lung fibrogenesis eludes us, and interventions capable of reversing or halting disease progression are not available. Pulmonary fibrosis is characterized by the excessive expression and uncontrolled deposition of extracellular matrix (ECM) proteins resulting in erosion of the tissue structure. Initially considered an ‘end-stage’ process elicited after injury, these events are now considered pathogenic and are believed to contribute to the course of the disease. By interacting with integrins capable of signal transduction and by influencing tissue mechanics, ECM proteins modulate processes ranging from cell adhesion and migration to differentiation and growth factor expression. In doing so, ECM proteins help orchestrate complex developmental processes and maintain tissue homeostasis. However, poorly controlled deposition of ECM proteins promotes inflammation, fibroproliferation, and aberrant differentiation of cells, and has been implicated in the pathogenesis of pulmonary fibrosis, atherosclerosis and cancer. Considering their vital functions, ECM proteins are the target of investigation, and oxidation–reduction (redox) reactions have emerged as important regulators of the ECM. Oxidative stress invariably accompanies lung disease and promotes ECM expression directly or through the overproduction of pro-fibrotic growth factors, while affecting integrin binding and activation. In vitro and in vivo investigations point to redox reactions as targets for intervention in pulmonary fibrosis and related disorders, but studies in humans have been disappointing probably due to the narrow impact of the interventions tested, and our poor understanding of the factors that regulate these complex reactions. This review is not meant to

  15. Extracellular matrix components mark the territories of circumventricular organs.

    PubMed

    Pócsai, Károly; Kálmán, Mihály

    2014-04-30

    In the central nervous system the extracellular matrix has important roles, e.g. supporting the extracellular space, controlling the tissue hydration, binding soluble factors and influencing their diffusion. The distribution of the extracellular matrix components in the brain has been mapped but data on the circumventricular organs (CVOs) is not available yet. The CVOs lack the blood-brain barrier and have relatively large perivascular spaces. The present study investigates tenascin-R and the lecticans: aggrecan, brevican, neurocan, and versican in the median eminence, the area postrema, the vascular organ of the lamina terminalis, the subfornical organ, the pineal body and the subcommissural organ of the rat applying immunohistochemical methods, and lectin histochemistry, using Wisteria floribunda agglutinin (WFA). The extracellular matrix components were found intensely expressed in the CVOs with two exceptions: aggrecan immunoreactivity visualized only neurons in the arcuate nucleus, and the subcommissural organ was not labeled with either WFA, or lecticans, or tenascin-R. The different labelings usually overlapped each other. The distribution of the extracellular matrix components marked the territories of the CVOs. Considering these we suppose that the extracellular matrix is essential in the maintenance of CVO functions providing the large extracellular space which is required for diffusion and other processes important in their chemosensitive and neurosecretory activities. The decrease of extracellular matrix beyond the border of the organs may contribute to the control of the diffusion of molecules from the CVOs into the surrounding brain substance. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Random Matrix Theory of Rigidity in Soft Matter

    NASA Astrophysics Data System (ADS)

    Yamanaka, Masanori

    2015-06-01

    We study the rigidity or softness of soft matter using the characteristic scale of coupling formation developed in random matrix theory. The eigensystems of the timescale-dependent cross-correlation matrix, which are obtained from the time series data of the atomic coordinates of a protein produced by the all-atom molecular dynamics of the solvent, are analyzed. As an example, we present a result for a protein lysozyme, PDBID:1AKI. We find that there are at least three different time scales involved in the coupling formation of correlated sectors of atoms and at least two different time scales for the size of the correlated sectors. These five time scales coexist simultaneously. We compare the results with those of the normal mode analysis and find a crossover of the distribution of the dominant vibrational components.

  17. Effects of ionizing radiation on extracellular matrix

    NASA Astrophysics Data System (ADS)

    Mohamed, F.; Bradley, D. A.; Winlove, C. P.

    2007-09-01

    The extracellular matrix is a ubiquitous and important component of tissues. We investigated the effects of ionizing radiation on the physical properties of its principal macromolecular components, pericardial collagen, ligament elastin and hyaluronan, a representative glycosaminoglycan. Samples were exposed to X-rays from an electron linear accelerator in the range of 10-100 Gy to cover the range of irradiation exposure during radiotherapy. A uniaxial mechanical testing protocol was used to characterize the fibrous proteins. For pericardial tissue the major change was an increase in the elastic modulus in the toe region of the curve (⩽20% strain), from 23±18 kPa for controls to 57±22 kPa at a dose of 10 Gy ( p=0.01, α=0.05). At larger strain (⩾20% strain), the elastic modulus in the linear region decreased from 1.92±0.70 MPa for control pericardium tissue to 1.31±0.56 MPa ( p=0.01, α=0.05) for 10 Gy X-irradiated sample. Similar observations have been made previously on tendon collagen at larger strains. For elastin, the stress-strain relationship was linear up to 30% strain, but the elastic modulus decreased significantly with irradiation (controls 626±65 kPa, irradiated 474±121 kPa ( p=0.02, α=0.05), at 10 Gy X-irradiation). The results suggest that for collagen the primary effect of irradiation is generation of additional cross-links, while for elastin chain scissions are important. The viscosity of HA (at 1.25% w/v and 0.125% w/v) was measured by both cone and plate and capillary viscometry, the former providing measurement at uniform shear rate and the latter providing a more sensitive indication of changes at low viscosity. Both techniques revealed a dose-dependent reduction in viscosity (from 3400±194 cP for controls to 1500±88 cP at a shear rate of 2 s -1 and dose of 75 Gy), again suggesting depolymerization.

  18. The extracellular matrix of blood vessels.

    PubMed

    Eble, Johannes A; Niland, Stephan

    2009-01-01

    Blood vessels are highly organized and complex structure, which are far more than simple tubes conducting the blood to almost any tissue of the body. They are able to autonomously regulate the blood flow, thus providing the tissues an optimal support of oxygen and nutrients and an efficient removal of waste products. In higher organisms, the blood vessel forms a closed circuit system, which additionally has the ability to seal itself in case of leakage as a result of injury. The blood vessel system does not only transport soluble substances, but also serves as "highway" system for leukocytes to patrol the body during the immunological surveillance and to reach the inflammation site quickly. In a complex interplay with the vascular wall, leukocytes are able to penetrate the blood vessel without any obvious leakage. Pathologically, tumor cells subvert the blood vessel system to disseminate from the primary tumor and colonize distant organs during metastasis. The extracellular matrix (ECM) of a blood vessel contributes substantially to the diverse functions of the blood vessel. First, the ECM constitutes the scaffold which keeps the histological structure of the vessel wall in shape but also bears the enormous and permanent mechanical forces levied on the vessel by the pulsatile blood flow in the arteries and by vasoconstriction, which regulates blood flow and pressure. The complex network of elastic fibers and tensile forces-bearing networks are well adapted to accomplish these mechanical tasks. Second, the ECM provides informational cues to the vascular cells, thus regulating their proliferation and differentiation. Third, ECM molecules can store, mask, present or sequester growth factors, thereby modulating their effects remarkably. Furthermore, several ECM molecules serve additional functions within the blood vessel. Their expression is altered in a spatial and temporal pattern during blood vessel formation and remodeling. In contrast to vasculogenesis during

  19. Extracellular matrix as a driver for lung regeneration.

    PubMed

    Balestrini, Jenna L; Niklason, Laura E

    2015-03-01

    Extracellular matrix has manifold roles in tissue mechanics, guidance of cellular behavior, developmental biology, and regenerative medicine. Over the past several decades, various pre-clinical and clinical studies have shown that many connective tissues may be replaced and/or regenerated using suitable extracellular matrix scaffolds. More recently, decellularization of lung tissue has shown that gentle removal of cells can leave behind a "footprint" within the matrix that may guide cellular adhesion, differentiation and homing following cellular repopulation. Fundamental issues like understanding matrix composition and micro-mechanics remain difficult to tackle, largely because of a lack of available assays and tools for systematically characterizing intact matrix from tissues and organs. This review will critically examine the role of engineered and native extracellular matrix in tissue and lung regeneration, and provide insights into directions for future research and translation.

  20. Extracellular Matrix Scaffolds for Tissue Engineering and Regenerative Medicine.

    PubMed

    Yi, Sheng; Ding, Fei; Gong, Leiiei; Gu, Xiaosong

    2017-01-01

    The extracellular matrix is produced by the resident cells in tissues and organs, and secreted into the surrounding medium to provide biophysical and biochemical support to the surrounding cells due to its content of diverse bioactive molecules. Recently, the extracellular matrix has been used as a promising approach for tissue engineering. Emerging studies demonstrate that extracellular matrix scaffolds are able to create a favorable regenerative microenvironment, promote tissue-specific remodeling, and act as an inductive template for the repair and functional reconstruction of skin, bone, nerve, heart, lung, liver, kidney, small intestine, and other organs. In the current review, we will provide a critical overview of the structure and function of various types of extracellular matrix, the construction of three-dimensional extracellular matrix scaffolds, and their tissue engineering applications, with a focus on translation of these novel tissue engineered products to the clinic. We will also present an outlook on future perspectives of the extracellular matrix in tissue engineering and regenerative medicine.

  1. Response of zonal chondrocytes to extracellular matrix-hydrogels.

    PubMed

    Hwang, Nathaniel S; Varghese, Shyni; Lee, H Janice; Theprungsirikul, Parnduangjai; Canver, Adam; Sharma, Blanka; Elisseeff, Jennifer

    2007-09-04

    We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses.

  2. RESPONSE OF ZONAL CHONDROCYTES TO EXTRACELLULAR MATRIX-HYDROGELS

    PubMed Central

    Hwang, Nathaniel S.; Varghese, Shyni; Lee, H. Janice; Theprungsirikul, Parnduangjai; Canver, Adam; Sharma, Blanka; Elisseeff, Jennifer

    2009-01-01

    We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses. PMID:17692846

  3. Increasing 3D Matrix Rigidity Strengthens Proliferation and Spheroid Development of Human Liver Cells in a Constant Growth Factor Environment.

    PubMed

    Bomo, Jérémy; Ezan, Frédéric; Tiaho, François; Bellamri, Medjda; Langouët, Sophie; Theret, Nathalie; Baffet, Georges

    2016-03-01

    Mechanical forces influence the growth and shape of virtually all tissues and organs. Recent studies show that increased cell contractibility, growth and differentiation might be normalized by modulating cell tensions. Particularly, the role of these tensions applied by the extracellular matrix during liver fibrosis could influence the hepatocarcinogenesis process. The objective of this study is to determine if 3D stiffness could influence growth and phenotype of normal and transformed hepatocytes and to integrate extracellular matrix (ECM) stiffness to tensional homeostasis. We have developed an appropriate 3D culture model: hepatic cells within three-dimensional collagen matrices with varying rigidity. Our results demonstrate that the rigidity influenced the cell phenotype and induced spheroid clusters development whereas in soft matrices, Huh7 transformed cells were less proliferative, well-spread and flattened. We confirmed that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas ERK2 mainly controlled proliferation. As compared to 2D culture, 3D cultures are associated with epithelial markers expression. Interestingly, proliferation of normal hepatocytes was also induced in rigid gels. Furthermore, biotransformation activities are increased in 3D gels, where CYP1A2 enzyme can be highly induced/activated in primary culture of human hepatocytes embedded in the matrix. In conclusion, we demonstrated that increasing 3D rigidity could promote proliferation and spheroid developments of liver cells demonstrating that 3D collagen gels are an attractive tool for studying rigidity-dependent homeostasis of the liver cells embedded in the matrix and should be privileged for both chronic toxicological and pharmacological drug screening. © 2015 Wiley Periodicals, Inc.

  4. The extracellular matrix modulates the hallmarks of cancer.

    PubMed

    Pickup, Michael W; Mouw, Janna K; Weaver, Valerie M

    2014-12-01

    The extracellular matrix regulates tissue development and homeostasis, and its dysregulation contributes to neoplastic progression. The extracellular matrix serves not only as the scaffold upon which tissues are organized but provides critical biochemical and biomechanical cues that direct cell growth, survival, migration and differentiation and modulate vascular development and immune function. Thus, while genetic modifications in tumor cells undoubtedly initiate and drive malignancy, cancer progresses within a dynamically evolving extracellular matrix that modulates virtually every behavioral facet of the tumor cells and cancer-associated stromal cells. Hanahan and Weinberg defined the hallmarks of cancer to encompass key biological capabilities that are acquired and essential for the development, growth and dissemination of all human cancers. These capabilities include sustained proliferation, evasion of growth suppression, death resistance, replicative immortality, induced angiogenesis, initiation of invasion, dysregulation of cellular energetics, avoidance of immune destruction and chronic inflammation. Here, we argue that biophysical and biochemical cues from the tumor-associated extracellular matrix influence each of these cancer hallmarks and are therefore critical for malignancy. We suggest that the success of cancer prevention and therapy programs requires an intimate understanding of the reciprocal feedback between the evolving extracellular matrix, the tumor cells and its cancer-associated cellular stroma.

  5. Monitoring of Extracellular Matrix Formation using Nanosecond Pulsed Laser

    NASA Astrophysics Data System (ADS)

    Ishihara, Miya; Sato, Masato; Mitani, Genya; Nagai, Toshihiro; Kutsuna, Toshiharu; Mochida, Joji; Kikuchi, Makoto

    There is a new demand in the field of tissue engineering for evaluation technology of extracellular matrix because the extracellular matrix plays an important role in the function of skeletal tissue such as articular cartilage. We previously proposed a noninvasive method of viscoelastic characterization of tissue phantom, based on the photoacoustic measurement. The purpose of this study was to verify the applicability of the photoacoustic measurement method for monitoring of the development of extracellular matrix using tissue engineering technology. The decay times measured by the photoacoustic method were varied with culture periods when tissue-engineered articular cartilages with various culture periods (-12 weeks) were used as samples. Tissue-engineered cartilage cultured for a long period showed shorter decay times, indicating that the samples approached an elastic solid from a rheological viewpoint. By comparison between biochemical analyses and biomechanical studies, we proved that the photoacoustic signal was a good indicator for evaluating extracellular matrix formation because the change of the photoacoustic decay times would reflect the production of an extracellular matrix.

  6. Cross-linked matrix rigidity and soluble retinoids synergize in nuclear lamina regulation of stem cell differentiation

    PubMed Central

    Ivanovska, Irena L.; Swift, Joe; Spinler, Kyle; Dingal, Dave; Cho, Sangkyun; Discher, Dennis E.

    2017-01-01

    Synergistic cues from extracellular matrix and soluble factors are often obscure in differentiation. Here the rigidity of cross-linked collagen synergizes with retinoids in the osteogenesis of human marrow mesenchymal stem cells (MSCs). Collagen nanofilms serve as a model matrix that MSCs can easily deform unless the film is enzymatically cross-linked, which promotes the spreading of cells and the stiffening of nuclei as both actomyosin assembly and nucleoskeletal lamin-A increase. Expression of lamin-A is known to be controlled by retinoic acid receptor (RAR) transcription factors, but soft matrix prevents any response to any retinoids. Rigid matrix is needed to induce rapid nuclear accumulation of the RARG isoform and for RARG-specific antagonist to increase or maintain expression of lamin-A as well as for RARG-agonist to repress expression. A progerin allele of lamin-A is regulated in the same manner in iPSC-derived MSCs. Rigid matrices are further required for eventual expression of osteogenic markers, and RARG-antagonist strongly drives lamin-A–dependent osteogenesis on rigid substrates, with pretreated xenografts calcifying in vivo to a similar extent as native bone. Proteomics-detected targets of mechanosensitive lamin-A and retinoids underscore the convergent synergy of insoluble and soluble cues in differentiation. PMID:28566555

  7. Formation of extracellular matrix by cultured rat mesangial cells.

    PubMed Central

    Ishimura, E.; Sterzel, R. B.; Budde, K.; Kashgarian, M.

    1989-01-01

    Formation of extracellular matrix (ECM) by mesangial cells (MCs) contributes to progressive glomerulosclerosis. The authors investigated the production and distribution of ECM constituents by cultured rat MCs, using immunocytochemistry and immunoelectron microscopy. Staining for all ECM constituents increased after serum feeding. Localization was strictly intracellular until confluency, when extracellular deposition of collagen IV and laminin appeared, followed by fibronectin and collagen III. In parallel, the intracellular staining for these proteins diminished markedly. Neither extracellular deposition nor intracellular loss was observed for collagen I and thrombospondin. On surfaces coated with collagen IV or laminin, extracellular deposition of ECM constituents clearly preceded confluency. These results indicate that synthesis of ECM constituents parallels MC growth, and that extracellular deposition of ECM occurs at cell-cell contact. Collagen IV or laminin secreted by MCs in the substratum accelerates production and facilitates secretion of other ECM constituents in an autocrine fashion. Images Figure 1 Figure 3 Figure 5 Figure 6 Figure 8 PMID:2650558

  8. Vitamin A Deficiency and Alterations in the Extracellular Matrix

    PubMed Central

    Barber, Teresa; Esteban-Pretel, Guillermo; Marín, María Pilar; Timoneda, Joaquín

    2014-01-01

    Vitamin A or retinol which is the natural precursor of several biologically active metabolites can be considered the most multifunctional vitamin in mammals. Its deficiency is currently, along with protein malnutrition, the most serious and common nutritional disorder worldwide. It is necessary for normal embryonic development and postnatal tissue homeostasis, and exerts important effects on cell proliferation, differentiation and apoptosis. These actions are produced mainly by regulating the expression of a variety of proteins through transcriptional and non-transcriptional mechanisms. Extracellular matrix proteins are among those whose synthesis is known to be modulated by vitamin A. Retinoic acid, the main biologically active form of vitamin A, influences the expression of collagens, laminins, entactin, fibronectin, elastin and proteoglycans, which are the major components of the extracellular matrix. Consequently, the structure and macromolecular composition of this extracellular compartment is profoundly altered as a result of vitamin A deficiency. As cell behavior, differentiation and apoptosis, and tissue mechanics are influenced by the extracellular matrix, its modifications potentially compromise organ function and may lead to disease. This review focuses on the effects of lack of vitamin A in the extracellular matrix of several organs and discusses possible molecular mechanisms and pathologic implications. PMID:25389900

  9. A Transitional Extracellular Matrix Instructs Cell Behavior During Muscle Regeneration

    PubMed Central

    Odelberg, Shannon J.; Simon, Hans-Georg

    2011-01-01

    Urodele amphibians regenerate appendages through the recruitment of progenitor cells into a blastema that rebuilds the lost tissue. Blastemal formation is accompanied by extensive remodeling of the extracellular matrix. Although this remodeling process is important for appendage regeneration, it is not known whether the remodeled matrix directly influences the generation and behavior of blastemal progenitor cells. By integrating in vivo 3-dimensional spatiotemporal matrix maps with in vitro functional time-lapse imaging, we show that key components of this dynamic matrix, hyaluronic acid, tenascin-C and fibronectin, differentially direct cellular behaviors including DNA synthesis, migration, myotube fragmentation and myoblast fusion. These data indicate that both satellite cells and fragmenting myofibers contribute to the regeneration blastema and that the local extracellular environment provides instructive cues for the regenerative process. The fact that amphibian and mammalian myoblasts exhibit similar responses to various matrices suggests that the ability to sense and respond to regenerative signals is evolutionarily conserved. PMID:20478295

  10. Extracellular Matrix Assembly in Diatoms (Bacillariophyceae)1

    PubMed Central

    Wustman, Brandon A.; Lind, Jan; Wetherbee, Richard; Gretz, Michael R.

    1998-01-01

    Achnanthes longipes is a marine, biofouling diatom that adheres to surfaces via adhesive polymers extruded during motility or organized into structures called stalks that contain three distinct regions: the pad, shaft, and collar. Four monoclonal antibodies (AL.C1–AL.C4) and antibodies from two uncloned hybridomas (AL.E1 and AL.E2) were raised against the extracellular adhesives of A. longipes. Antibodies were screened against a hot-water-insoluble/hot-bicarbonate-soluble-fraction. The hot-water-insoluble/hot-bicarbonate-soluble fraction was fractionated to yield polymers in three size ranges: F1, ≥ 20,000,000 Mr; F2, ≅100,000 Mr; and F3, <10,000 Mr relative to dextran standards. The ≅100,000-Mr fraction consisted of highly sulfated (approximately 11%) fucoglucuronogalactans (FGGs) and low-sulfate (approximately 2%) FGGs, whereas F1 was composed of O-linked FGG (F2)-polypeptide (F3) complexes. AL.C1, AL.C2, AL.C4, AL.E1, and AL.E2 recognized carbohydrate complementary regions on FGGs, with antigenicity dependent on fucosyl-containing side chains. AL.C3 was unique in that it had a lower affinity for FGGs and did not label any portion of the shaft. Enzyme-linked immunosorbent assay and immunocytochemistry indicated that low-sulfate FGGs are expelled from pores surrounding the raphe terminus, creating the cylindrical outer layers of the shaft, and that highly sulfated FGGs are extruded from the raphe, forming the central core. Antibody-labeling patterns and other evidence indicated that the shaft central-core region is related to material exuded from the raphe during cell motility. PMID:9536061

  11. A Look inside the Listeria monocytogenes Biofilms Extracellular Matrix

    PubMed Central

    Colagiorgi, Angelo; Di Ciccio, Pierluigi; Zanardi, Emanuela; Ghidini, Sergio; Ianieri, Adriana

    2016-01-01

    Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS’s). In this review, we summarized recent efforts performed in order to better characterize the polymeric substances that compose the extracellular matrix (ECM) of L. monocytogenes biofilms. EPS extraction and analysis led to the identification of polysaccharides, proteins, extracellular DNA, and other molecules within the listerial ECM. All this knowledge will be useful for increasing food protection, suggesting effective strategies for the minimization of persistence of L. monocytogenes in food industry environments. PMID:27681916

  12. Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

    NASA Technical Reports Server (NTRS)

    Globus. Ruth K.; Almeida, Eduardo A. C.; Searby, Nancy D.; Bowley, Susan M. (Technical Monitor)

    2000-01-01

    Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

  13. Defects in extracellular matrix structural proteins in the osteochondrodysplasias.

    PubMed

    Cohn, D H

    2001-01-01

    Mutations in the genes that encode structural proteins of the extracellular matrix affect one or more steps in the diverse set of coordinated events necessary for ordered skeletal development. Depending on the role of the gene product and the severity of the defect, disruption of endochondral ossification and linear growth, the structural integrity and stability of articular cartilage, and/or mineralization can occur. Several themes have emerged from the molecular dissection of these disorders; most of the osteochondrodysplasias that result from defects in structural proteins are inherited in an autosomal dominant fashion; a spectrum of related clinical phenotypes can be produced by distinct mutations in the same gene; haploinsufficiency for the gene product usually produces a milder clinical phenotype than do mutations resulting in synthesis of structurally abnormal proteins. For structural defects, a dominant-negative effect resulting from presence of the abnormal protein in the matrix appears to be the primary determinant of phenotype. Secondary effects on extracellular matrix protein structure can result from defects in post-translational maturation, including hydroxylation, sulfation and proteolytic cleavage, and produce distinct osteochondrodysplasias. Overall, the inherited disorders of skeletogenesis have revealed the exquisite sensitivity of the architecture of the extracellular matrix to the quantity and quality of matrix molecules.

  14. Specialisation of extracellular matrix for function in tendons and ligaments.

    PubMed

    Birch, Helen L; Thorpe, Chavaunne T; Rumian, Adam P

    2013-01-01

    Tendons and ligaments are similar structures in terms of their composition, organisation and mechanical properties. The distinction between them stems from their anatomical location; tendons form a link between muscle and bone while ligaments link bones to bones. A range of overlapping functions can be assigned to tendon and ligaments and each structure has specific mechanical properties which appear to be suited for particular in vivo function. The extracellular matrix in tendon and ligament varies in accordance with function, providing appropriate mechanical properties. The most useful framework in which to consider extracellular matrix differences therefore is that of function rather than anatomical location. In this review we discuss what is known about the relationship between functional requirements, structural properties from molecular to gross level, cellular gene expression and matrix turnover. The relevance of this information is considered by reviewing clinical aspects of tendon and ligament repair and reconstructive procedures.

  15. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps

    PubMed Central

    Cabezas-Olcoz, Jonathan; Wang, Steven X.; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E.

    2016-01-01

    Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix. PMID:27622514

  16. Extracellular matrix components direct porcine muscle stem cell behavior

    SciTech Connect

    Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J.

    2010-02-01

    In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.

  17. LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation.

    PubMed

    Asai, Nobuyuki; Ohkawara, Bisei; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

    2014-08-22

    Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.

    PubMed

    Al Shammari, Basim; Shiomi, Takayuki; Tezera, Liku; Bielecka, Magdalena K; Workman, Victoria; Sathyamoorthy, Tarangini; Mauri, Francesco; Jayasinghe, Suwan N; Robertson, Brian D; D'Armiento, Jeanine; Friedland, Jon S; Elkington, Paul T

    2015-08-01

    A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Neuronal growth cones and regeneration: gridlock within the extracellular matrix

    PubMed Central

    Snow, Diane M.

    2014-01-01

    The extracellular matrix is a diverse composition of glycoproteins and proteoglycans found in all cellular systems. The extracellular matrix, abundant in the mammalian central nervous system, is temporally and spatially regulated and is a dynamic “living” entity that is reshaped and redesigned on a continuous basis in response to changing needs. Some modifications are adaptive and some are maladaptive. It is the maladaptive responses that pose a significant threat to successful axonal regeneration and/or sprouting following traumatic and spinal cord injuries, and has been the focus of a myriad of research laboratories for many years. This review focuses largely on the extracellular matrix component, chondroitin sulfate proteoglycans, with certain comparisons to heparan sulfate proteoglycans, which tend to serve opposite functions in the central nervous system. Although about equally as well characterized as some of the other proteoglycans such as hyaluronan and dermatan sulfate proteoglycan, chondroitin sulfate proteoglycans are the most widely researched and discussed proteoglycans in the field of axonal injury and regeneration. Four laboratories discuss various aspects of chondroitin sulfate proteoglycans and proteoglycans in general with respect to their structure and function (Beller and Snow), the recent discovery of specific chondroitin sulfate proteoglycan receptors and what this may mean for increased advancements in the field (Shen), extracellular matrix degradation by matrix metalloproteinases, which sculpt and resculpt to provide support for outgrowth, synapse formation, and synapse stability (Phillips et al.), and the perilesion microenvironment with respect to immune system function in response to proteoglycans and central nervous system injuries (Jakeman et al.). PMID:25206821

  20. Vascular wall extracellular matrix proteins and vascular diseases

    PubMed Central

    Xu, Junyan; Shi, Guo-Ping

    2014-01-01

    Extracellular matrix proteins form the basic structure of blood vessels. Along with providing basic structural support to blood vessels, matrix proteins interact with different sets of vascular cells via cell surface integrin or non-integrin receptors. Such interactions induce vascular cell de novo synthesis of new matrix proteins during blood vessel development or remodeling. Under pathological conditions, vascular matrix proteins undergo proteolytic processing, yielding bioactive fragments to influence vascular wall matrix remodeling. Vascular cells also produce alternatively spliced variants that induce vascular cell production of different matrix proteins to interrupt matrix homeostasis, leading to increased blood vessel stiffness; vascular cell migration, proliferation, or death; or vascular wall leakage and rupture. Destruction of vascular matrix proteins leads to vascular cell or blood-borne leukocyte accumulation, proliferation, and neointima formation within the vascular wall; blood vessels prone to uncontrolled enlargement during blood flow diastole; tortuous vein development; and neovascularization from existing pathological tissue microvessels. Here we summarize discoveries related to blood vessel matrix proteins within the past decade from basic and clinical studies in humans and animals — from expression to cross-linking, assembly, and degradation under physiological and vascular pathological conditions, including atherosclerosis, aortic aneurysms, varicose veins, and hypertension. PMID:25045854

  1. Gene evolution and functions of extracellular matrix proteins in teeth

    PubMed Central

    Yoshizaki, Keigo; Yamada, Yoshihiko

    2013-01-01

    The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the “core matrisome” in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfect (OI) and amelogenesis imperfect (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins. PMID:23539364

  2. Regulation of extracellular matrix biosynthesis by matrix components

    SciTech Connect

    Holderbaum, D.; Ehrhart, L.A.

    1986-03-01

    The authors have previously shown that smooth muscle cells derived from healthy rabbit aortic media synthesize less collagen and fibronectin when grown on culture dishes coated with rabbit plasma fibronectin. In these cultures noncollagen protein synthesis was not affected, suggesting a specific regulatory mechanism. Their current studies expand this observation by examining the ability of proteolytically derived, specific domains of plasma fibronectin to effect decreases in collagen and fibronectin synthesis by cultured arterial smooth muscle. Rabbit plasma fibronectin was digested with bovine ..cap alpha..-chymotrypsin by the method of Hahn and Yamada. The resultant proteolytic fragments were separated by their ability to bind to gelatin-agarose. Culture dishes were coated with either (1) cell binding fragment of fibronectin, (2) gelatin binding fragment, (3) intact fibronectin, (4) type I collagen derived from lathyritic rat skin or (5) bovine serum albumin. Preconfluent cultures were labeled with /sup 3/H-Pro for 24 hr. Fibronectin synthesis was determined by immunoprecipitation of /sup 3/H-fibronectin. Collagen synthesis was measured by monitoring /sup 3/H-Hyp formation. Decreased collagen and fibronectin synthesis was evident in cells grown on intact fibronectin, cell binding fragment of fibronectin and type I collagen. Cells plated on gelatin binding fragment synthesized both collagen and fibronectin at levels comparable to cells on albumin coated dishes. They conclude that the regulatory activity of fibronectin on matrix biosynthesis resides on the cell binding domain of the molecule and that type I collagen can exert a similar effect.

  3. Extracellular matrix mediates epithelial effects on chondrogenesis in vitro.

    PubMed

    Solursh, M; Jensen, K L; Zanetti, N C; Linsenmayer, T F; Reiter, R S

    1984-10-01

    It has been previously observed that single chick embryonic limb mesenchymal cells can differentiate into chondrocytes without cell-cell interactions when cultured in collagen or agarose gels. In the present study, limb ectoderm, but not dermis, inhibits chondrogenesis when placed on such collagen gel cultures. The inhibitory influence can be transmitted extensive distances in the gel, even when the ectoderm is placed on a porous filter. Collagen gels, preconditioned with limb ectoderms, are also inhibitory to chondrogenesis. On the other hand, chondrogenesis is less inhibited by ectoderm when the mesenchymal cells are placed in agarose. These results suggest that the antichondrogenic effect of limb ectoderm is mediated through alterations of the collagenous extracellular matrix and support the idea that the extracellular matrix must be considered as an organized, functional unit capable of regulating cell differentiation.

  4. Evolution of cell interactions with extracellular matrix during carcinogenesis.

    PubMed

    Alexandrova, A Y

    2008-07-01

    Interaction of cells with extracellular matrix (ECM) largely defines migration capacity of cells and ways of their dissemination in normal tissue processes and during tumor progression. We review current knowledge about structure of cell adhesions with ECM and their alterations during carcinogenesis. We analyze how changes in structure of cell-matrix adhesions and ECM itself lead to acquisition of neoplastic properties by cells. Modern concepts of tumor cell motility and changes in the relationships of cells with ECM during tumor development are presented. Contemporary approaches for influencing the cell-ECM adhesion structures for inhibition of invasion and metastasis are briefly discussed.

  5. Effects of extracellular matrix on the malignant phenotype.

    PubMed Central

    Luikart, S. D.

    1988-01-01

    Extracellular matrix molecules, including collagen, glycosaminoglycans (usually linked to a protein core as proteoglycan), elastin, and glycoproteins, influence the initiation and maintenance of differentiation of a variety of cell types. These molecules bind to the cell surface at specific sites and nonspecifically by electrostatic forces. Such interactions may alter the cell's response to growth and differentiation factors. After neoplastic transformation, most cells retain some dependence on these factors. This paper reviews the influence of matrix components on the phenotype of a variety of malignant cells and concludes that in vitro studies of malignant cell behavior require the utilization of an appropriate microenvironment. PMID:3284211

  6. Tendon Extracellular Matrix Alterations in Ullrich Congenital Muscular Dystrophy

    PubMed Central

    Sardone, Francesca; Traina, Francesco; Bondi, Alice; Merlini, Luciano; Santi, Spartaco; Maraldi, Nadir Mario; Faldini, Cesare; Sabatelli, Patrizia

    2016-01-01

    Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient’s tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient’s tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient’s tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies. PMID:27375477

  7. Extracellular matrix stiffness and architecture govern intracellular rheology in cancer.

    PubMed

    Baker, Erin L; Bonnecaze, Roger T; Zaman, Muhammad H

    2009-08-19

    Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating ECM stiffness does not significantly affect the intracellular mechanical state when cells are attached to two-dimensional (2D) matrices. Switching from 2D to 3D matrices induces an order-of-magnitude shift in intracellular effective creep compliance and apparent elastic modulus. However, for a given matrix stiffness, partial blocking of beta1 integrins mitigates the shift in intracellular mechanical state that is invoked by switching from a 2D to 3D matrix architecture. This finding suggests that the increased cell-matrix engagement inherent to a 3D matrix architecture may contribute to differences observed in viscoelastic properties between cells attached to 2D matrices and cells embedded within 3D matrices. In total, our observations show that ECM stiffness and architecture can strongly influence the intracellular mechanical state of cancer cells.

  8. Tendon Extracellular Matrix Alterations in Ullrich Congenital Muscular Dystrophy.

    PubMed

    Sardone, Francesca; Traina, Francesco; Bondi, Alice; Merlini, Luciano; Santi, Spartaco; Maraldi, Nadir Mario; Faldini, Cesare; Sabatelli, Patrizia

    2016-01-01

    Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient's tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient's tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient's tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies.

  9. Conformal Nanopatterning of Extracellular Matrix Proteins onto Topographically Complex Surfaces

    PubMed Central

    Sun, Yan; Jallerat, Quentin; Szymanski, John M.

    2015-01-01

    We report a method for conformal nanopatterning of extracellular matrix proteins onto engineered surfaces independent of underlying microtopography. This enables fibronectin, laminin, and other proteins to be applied to biomaterial surfaces in complex geometries inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface, used here to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment. PMID:25506720

  10. Matrix Rigidity Differentially Regulates Invadopodia Activity Through ROCK1 and ROCK2

    PubMed Central

    Jerrell, Rachel J.; Parekh, Aron

    2016-01-01

    ROCK activity increases due to ECM rigidity in the tumor microenvironment and promotes a malignant phenotype via actomyosin contractility. Invasive migration is facilitated by actin-rich adhesive protrusions known as invadopodia that degrade the ECM. Invadopodia activity is dependent on matrix rigidity and contractile forces suggesting that mechanical factors may regulate these subcellular structures through ROCK-dependent actomyosin contractility. However, emerging evidence indicates that the ROCK1 and ROCK2 isoforms perform different functions in cells suggesting that alternative mechanisms may potentially regulate rigidity-dependent invadopodia activity. In this study, we found that matrix rigidity drives ROCK signaling in cancer cells but that ROCK1 and ROCK2 differentially regulate invadopodia activity through separate signaling pathways via contractile (NM II) and non-contractile (LIMK) mechanisms. These data suggest that the mechanical rigidity of the tumor microenvironment may drive ROCK signaling through distinct pathways to enhance the invasive migration required for cancer progression and metastasis. PMID:26826790

  11. Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis.

    PubMed

    Hielscher, Abigail; Ellis, Kim; Qiu, Connie; Porterfield, Josh; Gerecht, Sharon

    2016-01-01

    The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis.

  12. Starved epithelial cells uptake extracellular matrix for survival

    PubMed Central

    Muranen, Taru; Iwanicki, Marcin P.; Curry, Natasha L.; Hwang, Julie; DuBois, Cory D.; Coloff, Jonathan L.; Hitchcock, Daniel S.; Clish, Clary B.; Brugge, Joan S.; Kalaany, Nada Y.

    2017-01-01

    Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize β4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell β4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition. PMID:28071763

  13. The role of extracellular matrix in spinal cord development.

    PubMed

    Wiese, Stefan; Faissner, Andreas

    2015-12-01

    The development of the spinal cord represents one of the most complex structure developments of the central nervous system (CNS) as it has to unfold along the longitudinal axis and within segmental cues. There it has to cope with on the one hand connection to the periphery (skeletal muscle, dermomyotome, smooth muscles) and connect it to the higher midbrain and cortical regions of the CNS. Major studies have been performed to analyze the specific subset of transcription factors of the different types of cells within the different segments of the spinal cord. But transcription factor expression is always a result of cellular positioning as the environment defines the intracellular changes during differentiation and in adulthood. The surrounding composed of mainly extracellular matrix does not only provide a "glue" to attach cells to each other but also provides signals with special domains docking to cell surface receptors and presents soluble molecules such as basic fibroblast growth factors (bFGFs) or Wnt-proteins. The availability of these molecules depends on the matrix composition and influences the transcription factor code of each cell. Recent research has also provided strong evidence that depletion of single matrix molecules like Tenascin C (TnC) can lead to developmental changes within the progenitor pools. Therefore beyond the transcription factor code that defines cellular properties we want to focus on the role of the extracellular matrix in the development of the spinal cord.

  14. Extracellular matrix structure governs invasion resistance in bacterial biofilms

    PubMed Central

    Nadell, Carey D; Drescher, Knut; Wingreen, Ned S; Bassler, Bonnie L

    2015-01-01

    Many bacteria are highly adapted for life in communities, or biofilms. A defining feature of biofilms is the production of extracellular matrix that binds cells together. The biofilm matrix provides numerous fitness benefits, including protection from environmental stresses and enhanced nutrient availability. Here we investigate defense against biofilm invasion using the model bacterium Vibrio cholerae. We demonstrate that immotile cells, including those identical to the biofilm resident strain, are completely excluded from entry into resident biofilms. Motile cells can colonize and grow on the biofilm exterior, but are readily removed by shear forces. Protection from invasion into the biofilm interior is mediated by the secreted protein RbmA, which binds mother–daughter cell pairs to each other and to polysaccharide components of the matrix. RbmA, and the invasion protection it confers, strongly localize to the cell lineages that produce it. PMID:25603396

  15. Extracellular matrix structure governs invasion resistance in bacterial biofilms.

    PubMed

    Nadell, Carey D; Drescher, Knut; Wingreen, Ned S; Bassler, Bonnie L

    2015-08-01

    Many bacteria are highly adapted for life in communities, or biofilms. A defining feature of biofilms is the production of extracellular matrix that binds cells together. The biofilm matrix provides numerous fitness benefits, including protection from environmental stresses and enhanced nutrient availability. Here we investigate defense against biofilm invasion using the model bacterium Vibrio cholerae. We demonstrate that immotile cells, including those identical to the biofilm resident strain, are completely excluded from entry into resident biofilms. Motile cells can colonize and grow on the biofilm exterior, but are readily removed by shear forces. Protection from invasion into the biofilm interior is mediated by the secreted protein RbmA, which binds mother-daughter cell pairs to each other and to polysaccharide components of the matrix. RbmA, and the invasion protection it confers, strongly localize to the cell lineages that produce it.

  16. Applying Proteomics to Investigate Extracellular Matrix in Health and Disease.

    PubMed

    Randles, Michael; Lennon, Rachel

    2015-01-01

    The molecular composition of basement membranes (BMs) has traditionally been investigated by candidate-based approaches leading to the identification of key structural components as described in previous chapters. Laminins, collagen IV, nidogens, perlecan, and type XV/XVIII collagen are integral to BMs with isoforms showing tissue specificity. More recently the application of mass spectrometry (MS)-based proteomics has led to the discovery of many more structural and regulatory components of BMs and more broadly, extracellular matrix (ECM). These investigations have revealed tissue-specific signatures of between 100 and 150 ECM components, demonstrating the complexity of the extracellular niche. In addition to providing a structural scaffold for cells, ECM is a dynamic extracellular environment capable of regulating the physical properties of tissues. Global investigations of ECM with proteomics in turn enable systems level analyses and when applied to health and disease states these investigations provide insights into pathways regulating matrix dysregulation. This chapter focuses on the methods used to extract ECM and on the analysis of its composition using MS-based proteomics, and it provides examples of how these approaches have been used to investigate health and disease states.

  17. Astrocytes and extracellular matrix in extrasynaptic volume transmission

    PubMed Central

    Vargová, Lýdia; Syková, Eva

    2014-01-01

    Volume transmission is a form of intercellular communication that does not require synapses; it is based on the diffusion of neuroactive substances across the brain extracellular space (ECS) and their binding to extrasynaptic high-affinity receptors on neurons or glia. Extracellular diffusion is restricted by the limited volume of the ECS, which is described by the ECS volume fraction α, and the presence of diffusion barriers, reflected by tortuosity λ, that are created, for example, by fine astrocytic processes or extracellular matrix (ECM) molecules. Organized astrocytic processes, ECM scaffolds or myelin sheets channel the extracellular diffusion so that it is facilitated in a certain direction, i.e. anisotropic. The diffusion properties of the ECS are profoundly influenced by various processes such as the swelling and morphological rebuilding of astrocytes during either transient or persisting physiological or pathological states, or the remodelling of the ECM in tumorous or epileptogenic tissue, during Alzheimer's disease, after enzymatic treatment or in transgenic animals. The changing diffusion properties of the ECM influence neuron–glia interaction, learning abilities, the extent of neuronal damage and even cell migration. From a clinical point of view, diffusion parameter changes occurring during pathological states could be important for diagnosis, drug delivery and treatment. PMID:25225101

  18. Production of extracellular polysaccharide matrix by Zoogloea ramigera.

    PubMed

    Parsons, A B; Dugan, P R

    1971-04-01

    Zoogloea ramigera 115 synthesized large amounts of matrix polymer from fructose, galactose, glucose, lactose, mannose, soluble starch, and sucrose when these carbohydrates were used as supplements to a chemically defined medium. All of them supported polymer synthesis to the extent that cultures thickened to a gel. Concentration of carbohydrate nutrients in the range 0.5 to 2.0% was not a critical factor in determining eventual total thickening to a gel, except in relation to the incubation time required. Glucose disappeared from the growth medium rapidly and correlated with increasing cell growth and poly-beta-hydroxybutyrate (PHB) accumulation. PHB concentration decreased as extracellular polymer was synthesized, suggesting a link between PHB and extracellular polymer production.

  19. Production of Extracellular Polysaccharide Matrix by Zoogloea ramigera

    PubMed Central

    Parsons, Alice B.; Dugan, Patrick R.

    1971-01-01

    Zoogloea ramigera 115 synthesized large amounts of matrix polymer from fructose, galactose, glucose, lactose, mannose, soluble starch, and sucrose when these carbohydrates were used as supplements to a chemically defined medium. All of them supported polymer synthesis to the extent that cultures thickened to a gel. Concentration of carbohydrate nutrients in the range 0.5 to 2.0% was not a critical factor in determining eventual total thickening to a gel, except in relation to the incubation time required. Glucose disappeared from the growth medium rapidly and correlated with increasing cell growth and poly-β-hydroxybutyrate (PHB) accumulation. PHB concentration decreased as extracellular polymer was synthesized, suggesting a link between PHB and extracellular polymer production. PMID:5575568

  20. Zinc deprivation inhibits extracellular matrix calcification through decreased synthesis of matrix proteins in osteoblasts.

    PubMed

    Alcantara, Ethel H; Lomeda, Ria-Ann R; Feldmann, Joerg; Nixon, Graeme F; Beattie, John H; Kwun, In-Sook

    2011-10-01

    Zinc is implicated as an activator for bone formation, however, its influence on bone calcification has not been reported. This study examined how zinc regulates the bone matrix calcification in osteoblasts. Two osteoblastic MC3T3-E1 cell subclones (SC 4 and SC 24 as high and low osteogenic differentiation, respectively) were cultured in normal osteogenic (OSM), Zinc deficient (Zn-, 1 μM), or adequate (Zn+, 15 μM) media up to 20 days. Cells (SC 4) were also supplemented with (50 μg/mL) or no ascorbic acid (AA) in combination with Zinc treatment. Zn- decreased collagen synthesis and matrix accumulation. Although AA is essential for collagen formation, its supplementation could not compensate for Zinc deficiency-induced detrimental effects on extracellular matrix mineralization. Zn- also decreased the medium and cell layer alkaline phosphatase ALP activity. This decreased ALP activity might cause the decrease of Pi accumulation in response to Zn-, as measured by von Kossa staining. Ca deposition in cell layers, measured by Alizarin red S staining, was also decreased by Zn(-) . Our findings suggest that zinc deprivation inhibits extracellular matrix calcification in osteoblasts by decreasing the synthesis and activity of matrix proteins, type I collagen and ALP, and decreasing Ca and Pi accumulation. Therefore zinc deficiency can be considered as risk factor for poor extracellular matrix calcification. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Structure and function of the skeletal muscle extracellular matrix.

    PubMed

    Gillies, Allison R; Lieber, Richard L

    2011-09-01

    The skeletal muscle extracellular matrix (ECM) plays an important role in muscle fiber force transmission, maintenance, and repair. In both injured and diseased states, ECM adapts dramatically, a property that has clinical manifestations and alters muscle function. Here we review the structure, composition, and mechanical properties of skeletal muscle ECM; describe the cells that contribute to the maintenance of the ECM; and, finally, overview changes that occur with pathology. New scanning electron micrographs of ECM structure are also presented with hypotheses about ECM structure–function relationships. Detailed structure–function relationships of the ECM have yet to be defined and, as a result, we propose areas for future study.

  2. Structure and Function of the Stratum Corneum Extracellular Matrix

    PubMed Central

    Elias, Peter M.

    2013-01-01

    The stratum corneum (SC) extracellular matrix (ECM) is enriched in lipids that are organized into lamellar bilayers, whose molecular architecture is now known. Although these bilayers are important for the permeability barrier, the ECM contains not only lipids but also enzymes, structural proteins, and antimicrobial peptides that impact barrier function. Yet, how such diverse components affect barrier function remains largely unknown. Static models of the epidermis may not do justice to the ECM, which is metabolically active, as it changes both structure and function as it transits to the surface. PMID:22895445

  3. A fast and mild decellularization protocol for obtaining extracellular matrix.

    PubMed

    Mirzarafie, Ariana; Grainger, Rhian K; Thomas, Ben; Bains, William; Ustok, Fatma I; Lowe, Chris R

    2014-04-01

    Degradation of extracellular matrix (ECM) function with age is a major cause of loss of tissue function with age that we would wish to reverse. Tissue engineering to provide replacement tissue requires an ECM-mimicking scaffold for cell organization. The standard protocols for achieving this take 10 days and include steps that may change the protein structure of the ECM. Here we describe a much shorter protocol for decellularizing chicken muscle, skin, and tendon samples that achieves the same efficiency as the original protocol without protein cross-link interference. Our protocol can be completed in 72 hr.

  4. Structure and Function of the Skeletal Muscle Extracellular Matrix

    PubMed Central

    Gillies, Allison R.; Lieber, Richard L.

    2011-01-01

    The skeletal muscle extracellular matrix (ECM) plays an important role in muscle fiber force transmission, maintenance, and repair. In both injured and diseased states, ECM adapts dramatically, a property thathas clinical manifestations and alters muscle function. Here, we review the structure, composition, and mechanical properties of skeletal muscle ECM, describe the cells that contribute to the maintenance of the ECM and, finally, overview changes that occur with pathology. New scanning electron micrographs of ECM structure are also presented with hypotheses about ECM structure-function relationships. Detailed structure-function relationships of the ECM have yet to be defined and, as a result, we propose areas for future studies. PMID:21949456

  5. The design of reversible hydrogels to capture extracellular matrix dynamics

    NASA Astrophysics Data System (ADS)

    Rosales, Adrianne M.; Anseth, Kristi S.

    2016-02-01

    The extracellular matrix (ECM) is a dynamic environment that constantly provides physical and chemical cues to embedded cells. Much progress has been made in engineering hydrogels that can mimic the ECM, but hydrogel properties are, in general, static. To recapitulate the dynamic nature of the ECM, many reversible chemistries have been incorporated into hydrogels to regulate cell spreading, biochemical ligand presentation and matrix mechanics. For example, emerging trends include the use of molecular photoswitches or biomolecule hybridization to control polymer chain conformation, thereby enabling the modulation of the hydrogel between two states on demand. In addition, many non-covalent, dynamic chemical bonds have found increasing use as hydrogel crosslinkers or tethers for cell signalling molecules. These reversible chemistries will provide greater temporal control of adhered cell behaviour, and they allow for more advanced in vitro models and tissue-engineering scaffolds to direct cell fate.

  6. Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13

    SciTech Connect

    Sakurai, Atsuo; Okahashi, Nobuo; Maruyama, Fumito; Ooshima, Takashi; Hamada, Shigeyuki; Nakagawa, Ichiro

    2008-08-29

    Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis.

  7. Extracellular matrix and its receptors in Drosophila neural development

    PubMed Central

    Broadie, Kendal; Baumgartner, Stefan; Prokop, Andreas

    2011-01-01

    Extracellular matrix (ECM) and matrix receptors are intimately involved in most biological processes. The ECM plays fundamental developmental and physiological roles in health and disease, including processes underlying the development, maintenance and regeneration of the nervous system. To understand the principles of ECM-mediated functions in the nervous system, genetic model organisms like Drosophila provide simple, malleable and powerful experimental platforms. This article provides an overview of ECM proteins and receptors in Drosophila. It then focuses on their roles during three progressive phases of neural development: 1) neural progenitor proliferation, 2) axonal growth and pathfinding and 3) synapse formation and function. Each section highlights known ECM and ECM-receptor components and recent studies done in mutant conditions to reveal their in vivo functions, all illustrating the enormous opportunities provided when merging work on the nervous system with systematic research into ECM-related gene functions. PMID:21688401

  8. The avian eggshell extracellular matrix as a model for biomineralization.

    PubMed

    Carrino, D A; Dennis, J E; Wu, T M; Arias, J L; Fernandez, M S; Rodriguez, J P; Fink, D J; Heuer, A H; Caplan, A I

    1996-01-01

    The avian eggshell is a complex, extracellularly assembled structure which contains both mineralized and non-mineralized regions. The composition of the hen eggshell organic matrix was examined by immunohistochemistry with antibodies to different extracellular matrix molecules. Type I collagen is found in the shell membranes, but only after treatment of the tissue sections with pepsin. When incomplete eggshells are removed from the oviduct and immunostained, type I collagen can be detected in the shell membranes without pepsin treatment. The shell membranes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of type X collagen in the shell membranes is surprising, since this collagen has not been found in any tissue other than hypertrophic cartilage. Immunostaining for various glycosaminoglycans shows the presence of keratan sulfate and dermatan sulfate. Several different antibodies to keratan sulfate stain different regions of the eggshell; one keratan sulfate epitope is prominent in the calcium reserve assemblies. Dermatan sulfate staining is very intense in the palisade region. Demineralized matrix from the palisade region was extracted with guanidine and fractionated by ion exchange chromatography. A approximately 200-kDa dermatan sulfate proteoglycan is found in these extracts, along with a number of protein components. This preparation was tested for its ability to affect calcium carbonate crystal formation in vitro. Pieces of demineralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteoglycan preparation were added to the solution from which the crystals were formed. This material causes a concentration-dependent change in crystal morphology to one in which the crystals are smaller and more rounded, which more closely approximates the crystals normally observed in eggshells. These results suggest

  9. The Extracellular Matrix In Development and Morphogenesis: A Dynamic View

    PubMed Central

    Rozario, Tania; DeSimone, Douglas W.

    2009-01-01

    The extracellular matrix (ECM) is synthesized and secreted by embryonic cells beginning at the earliest stages of development. Our understanding of ECM composition, structure and function has grown considerably in the last several decades and this knowledge has revealed that the extracellular microenvironment is critically important for cell growth, survival, differentiation and morphogenesis. ECM and the cellular receptors that interact with it mediate both physical linkages with the cytoskeleton and the bidirectional flow of information between the extracellular and intracellular compartments. This review considers the range of cell and tissue functions attributed to ECM molecules and summarizes recent findings specific to key developmental processes. The importance of ECM as a dynamic repository for growth factors is highlighted along with more recent studies implicating the 3-dimensional organization and physical properties of the ECM as it relates to cell signaling and the regulation of morphogenetic cell behaviors. Embryonic cell and tissue generated forces and mechanical signals arising from ECM adhesion represent emerging areas of interest in this field. PMID:19854168

  10. Specific Extracellular Matrix Remodeling Signature of Colon Hepatic Metastases

    PubMed Central

    Vezzio-Vie, Nadia; Bibeau, Frédéric; Ychou, Marc; Martineau, Pierre

    2013-01-01

    To identify genes implicated in metastatic colonization of the liver in colorectal cancer, we collected pairs of primary tumors and hepatic metastases before chemotherapy in 13 patients. We compared mRNA expression in the pairs of patients to identify genes deregulated during metastatic evolution. We then validated the identified genes using data obtained by different groups. The 33-gene signature was able to classify 87% of hepatic metastases, 98% of primary tumors, 97% of normal colon mucosa, and 95% of normal liver tissues in six datasets obtained using five different microarray platforms. The identified genes are specific to colon cancer and hepatic metastases since other metastatic locations and hepatic metastases originating from breast cancer were not classified by the signature. Gene Ontology term analysis showed that 50% of the genes are implicated in extracellular matrix remodeling, and more precisely in cell adhesion, extracellular matrix organization and angiogenesis. Because of the high efficiency of the signature to classify colon hepatic metastases, the identified genes represent promising targets to develop new therapies that will specifically affect hepatic metastasis microenvironment. PMID:24023955

  11. Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy.

    PubMed

    Edginton, Ryan S; Mattana, Sara; Caponi, Silvia; Fioretto, Daniele; Green, Ellen; Winlove, C Peter; Palombo, Francesca

    2016-09-15

    Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

  12. Extracellular matrix molecules as targets for brown spider venom toxins.

    PubMed

    Veiga, S S; Zanetti, V C; Braz, A; Mangili, O C; Gremski, W

    2001-07-01

    Loxoscelism, the term used to describe lesions and clinical manifestations induced by brown spider's venom (Loxosceles genus), has attracted much attention over the last years. Brown spider bites have been reported to cause a local and acute inflammatory reaction that may evolve to dermonecrosis (a hallmark of envenomation) and hemorrhage at the bite site, besides systemic manifestations such as thrombocytopenia, disseminated intravascular coagulation, hemolysis, and renal failure. The molecular mechanisms by which Loxosceles venoms induce injury are currently under investigation. In this review, we focused on the latest reports describing the biological and physiopathological aspects of loxoscelism, with reference mainly to the proteases recently described as metalloproteases and serine proteases, as well as on the proteolytic effects triggered by L. intermedia venom upon extracellular matrix constituents such as fibronectin, fibrinogen, entactin and heparan sulfate proteoglycan, besides the disruptive activity of the venom on Engelbreth-Holm-Swarm basement membranes. Degradation of these extracellular matrix molecules and the observed disruption of basement membranes could be related to deleterious activities of the venom such as loss of vessel and glomerular integrity and spreading of the venom toxins to underlying tissues.

  13. Mechanical model for a collagen fibril pair in extracellular matrix.

    PubMed

    Chan, Yue; Cox, Grant M; Haverkamp, Richard G; Hill, James M

    2009-04-01

    In this paper, we model the mechanics of a collagen pair in the connective tissue extracellular matrix that exists in abundance throughout animals, including the human body. This connective tissue comprises repeated units of two main structures, namely collagens as well as axial, parallel and regular anionic glycosaminoglycan between collagens. The collagen fibril can be modeled by Hooke's law whereas anionic glycosaminoglycan behaves more like a rubber-band rod and as such can be better modeled by the worm-like chain model. While both computer simulations and continuum mechanics models have been investigated for the behavior of this connective tissue typically, authors either assume a simple form of the molecular potential energy or entirely ignore the microscopic structure of the connective tissue. Here, we apply basic physical methodologies and simple applied mathematical modeling techniques to describe the collagen pair quantitatively. We found that the growth of fibrils was intimately related to the maximum length of the anionic glycosaminoglycan and the relative displacement of two adjacent fibrils, which in return was closely related to the effectiveness of anionic glycosaminoglycan in transmitting forces between fibrils. These reveal the importance of the anionic glycosaminoglycan in maintaining the structural shape of the connective tissue extracellular matrix and eventually the shape modulus of human tissues. We also found that some macroscopic properties, like the maximum molecular energy and the breaking fraction of the collagen, were also related to the microscopic characteristics of the anionic glycosaminoglycan.

  14. Mifepristone inhibits extracellular matrix formation in uterine leiomyoma.

    PubMed

    Patel, Amrita; Malik, Minnie; Britten, Joy; Cox, Jeris; Catherino, William H

    2016-04-01

    To characterize the efficacy of mifepristone treatment on extracellular matrix (ECM) production in leiomyomas. Laboratory study. University research laboratory. None. Treatment of human immortalized two-dimensional (2D) and three-dimensional (3D) leiomyoma and myometrial cells with mifepristone and the progestin promegestone (R5020). Expression of COL1A1, fibronectin, versican variant V0, and dermatopontin in treated leiomyoma cells by Western blot analysis and confirmatory immunohistochemistry staining of treated 3D cultures. Treatment with progestin stimulated production of COL1A1, fibronectin, versican, and dermatopontin. Mifepristone treatment inhibited protein production of these genes, most notably with versican expression. Combination treatment with both the agonist and antagonist further inhibited protein expression of these genes. Immunohistochemistry performed on 3D cultures demonstrated generalized inhibition of ECM protein concentration. Our study demonstrated that the progesterone agonist R5020 directly stimulated extracellular matrix components COL1A1, fibronectin, versican, and dermatopontin production in human leiomyoma cells. Progesterone antagonist mifepristone decreased protein production of these genes to levels comparable with untreated leiomyoma cells. Published by Elsevier Inc.

  15. Non-invasive multiphoton imaging of extracellular matrix structures

    PubMed Central

    Schenke-Layland, Katja

    2015-01-01

    Multiphoton microscopy has become a powerful method for the artifact-free, nondestructive evaluation of deep-tissue cells and extracellular matrix (ECM) structures in their native environment. By interacting with highly non-centrosymmetric molecular assemblies such as fibrillar collagen, the non-linear process called second harmonic generation (SHG) has also proven to be an important diagnostic tool for the visualization of ECM compartments in situ with submicron resolution without the need for tissue processing. This review reports on applications of multiphoton-induced autofluorescence and SHG microscopy to identify collagen and elastic fiber orientation in native, tissue-engineered and processed, as well as healthy and diseased, tissues and organs. SHG signal profiling was used to quantify ECM damage in various cardiovascular and exocrine tissues, as well as cartilage. These novel imaging modalities open the general possibility of high-resolution in situ and more important in vivo imaging of ECM structures, cells and intracellular organelles in living intact tissues. Heart valve leaflets have a unique extracellular matrix that is organized in distinct layers, which can be non-destructively visualized by multiphoton imaging. PMID:19343671

  16. The impact of aging on cardiac extracellular matrix.

    PubMed

    Meschiari, Cesar A; Ero, Osasere Kelvin; Pan, Haihui; Finkel, Toren; Lindsey, Merry L

    2017-02-01

    Age-related changes in cardiac homeostasis can be observed at the cellular, extracellular, and tissue levels. Progressive cardiomyocyte hypertrophy, inflammation, and the gradual development of cardiac fibrosis are hallmarks of cardiac aging. In the absence of a secondary insult such as hypertension, these changes are subtle and result in slight to moderate impaired myocardial function, particularly diastolic function. While collagen deposition and cross-linking increase during aging, extracellular matrix (ECM) degradation capacity also increases due to increased expression of matrix metalloproteinases (MMPs). Of the MMPs elevated with cardiac aging, MMP-9 has been extensively evaluated and its roles are reviewed here. In addition to proteolytic activity on ECM components, MMPs oversee cell signaling during the aging process by modulating cytokine, chemokine, growth factor, hormone, and angiogenic factor expression and activity. In association with elevated MMP-9, macrophage numbers increase in an age-dependent manner to regulate the ECM and angiogenic responses. Understanding the complexity of the molecular interactions between MMPs and the ECM in the context of aging may provide novel diagnostic indicators for the early detection of age-related fibrosis and cardiac dysfunction.

  17. Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

    PubMed Central

    Edginton, Ryan S.; Mattana, Sara; Caponi, Silvia; Fioretto, Daniele; Green, Ellen; Winlove, C. Peter; Palombo, Francesca

    2016-01-01

    Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis. PMID:27684584

  18. Using self-assembled monolayers to model the extracellular matrix.

    PubMed

    Mrksich, Milan

    2009-03-01

    The extracellular matrix is an insoluble aggregate of large proteins and glycosoaminoglycans that comprises the microenvironment of cells in tissue. The matrix displays a host of ligands that interact with cell-surface receptors to mediate the attachment and spreading of cells and regulate signaling processes. Studies of cell-matrix interactions and downstream signaling processes commonly employ substrates having an adsorbed layer of protein and are challenged by the difficulty in controlling the structure and activity of the immobilized protein. Significant effort has been directed towards the development of model substrates that present adhesion ligands in defined densities, orientations and environments. Among these approaches, self-assembled monolayers of alkanethiolates on gold offer a high level of control over the molecular structure of the surface and are well-suited to studies of cell adhesion. This review describes the design and use of monolayers for applications in cell biology, including the use of monolayers to evaluate the roles of peptide and protein ligands in cell-matrix interactions, the development of methods to pattern ligands on monolayers and applications to cell biology, the development of dynamic monolayers that can switch the activities of ligands presented to an adherent cell, and the rewiring of interactions between a cell and its substrate. These examples illustrate the flexibility inherent to monolayers for applications in cell biology.

  19. Extracellular matrix of the bovine ovarian membrana granulosa.

    PubMed

    Rodgers, R J; Irving Rodgers, H F

    2002-05-31

    Much is known about the control of the development of ovarian follicles by growth factors and hormones. The study of extracellular matrix in the ovary, though, is a relatively new area. To date much research has focused on identifying the matrix components present, and more recently, its production and the physiological roles. In this review we focus on the changes that occur in the follicular basal lamina from primordial follicles through to ovulation and formation of the corpus luteum, the changes that occur during follicular atresia, and we discuss our observations of a novel matrix which forms in the membrana granulosa. The follicular basal lamina changes considerably during follicular development in its expression pattern of type IV collagens. Of the laminin chains examined, there appears only to be an increase in amount, except for laminin alpha2. It is expressed only in a small proportion of healthy antral follicles and in the majority of atretic antral follicles. Call-Exner bodies have the same composition as the basal lamina, except they do not contain laminin alpha2, even when the follicular basal lamina does. The novel matrix that develops within the membrana granulosa is similar in composition to Call-Exner bodies which occur predominantly in preantral follicles, except that it is far more common in large antral follicles, does not induce polarization of the surrounding granulosa cells, and does not contain follicular fluid-like material as the Call-Exner bodies of some species do. The expression of this matrix occurs prior to and during the time when granulosa cells express steroidogenic enzymes. It does not exist in corpora lutea. In addition large luteal cells, derived from granulosa cells, do not appear to have a basal lamina. These findings suggest that the maturational changes in the membrana granulosa are accompanied by changes in the matrix.

  20. Modeling Extracellular Matrix Reorganization in 3D Environments

    PubMed Central

    Harjanto, Dewi; Zaman, Muhammad H.

    2013-01-01

    Extracellular matrix (ECM) remodeling is a key physiological process that occurs in a number of contexts, including cell migration, and is especially important for cellular form and function in three-dimensional (3D) matrices. However, there have been few attempts to computationally model how cells modify their environment in a manner that accounts for both cellular properties and the architecture of the surrounding ECM. To this end, we have developed and validated a novel model to simulate matrix remodeling that explicitly defines cells in a 3D collagenous matrix. In our simulation, cells can degrade, deposit, or pull on local fibers, depending on the fiber density around each cell. The cells can also move within the 3D matrix. Different cell phenotypes can be modeled by varying key cellular parameters. Using the model we have studied how two model cancer cell lines, of differing invasiveness, modify matrices with varying fiber density in their vicinity by tracking the metric of fraction of matrix occupied by fibers. Our results quantitatively demonstrate that in low density environments, cells deposit more collagen to uniformly increase fibril fraction. On the other hand, in higher density environments, the less invasive model cell line reduced the fibril fraction as compared to the highly invasive phenotype. These results show good qualitative and quantitative agreement with existing experimental literature. Our simulation is therefore able to function as a novel platform to provide new insights into the clinically relevant and physiologically critical process of matrix remodeling by helping identify critical parameters that dictate cellular behavior in complex native-like environments. PMID:23341900

  1. Invasion-promoting extracellular matrix composition enhances photodynamic therapy response in 3D pancreatic cancer models

    NASA Astrophysics Data System (ADS)

    Cramer, Gwendolyn M.; El-Hamidi, Hamid; Celli, Jonathan P.

    2017-02-01

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by extracellular matrix-rich stromal involvement, but it is not clear how ECM properties that affect invasiveness and chemotherapy response influence efficacy of photodynamic therapy (PDT). To disentangle the mechanical and biochemical effects of ECM composition, we measured the effects of various combinations of ECM proteins on growth behavior, invasive potential, and therapeutic response of multicellular 3D pancreatic tumor models. These spheroids were grown in attachment-free conditions before embedding in combinations of rheologically characterized collagen 1 and Matrigel combinations and treated with oxaliplatin chemotherapy and PDT. We find that cells invading from collagen-embedded tumor spheroids, the least rigid ECM substrate described here, displayed better response to PDT than to oxaliplatin chemotherapy. Overall, our results support that ECM-mediated invading PDAC populations remain responsive to PDT in conditions that induce chemoresistance.

  2. The Extracellular Matrix in Photosynthetic Mats: A Cyanobacterial Gingerbread House

    NASA Astrophysics Data System (ADS)

    Stuart, R.; Stannard, W.; Bebout, B.; Pett-Ridge, J.; Mayali, X.; Weber, P. K.; Lipton, M. S.; Lee, J.; Everroad, R. C.; Thelen, M.

    2014-12-01

    Hypersaline laminated cyanobacterial mats are excellent model systems for investigating photoautotrophic contributions to biogeochemical cycling on a millimeter scale. These self-sustaining ecosystems are characterized by steep physiochemical gradients that fluctuate dramatically on hour timescales, providing a dynamic environment to study microbial response. However, elucidating the distribution of energy from light absorption into biomass requires a complete understanding of the various constituents of the mat. Extracellular polymeric substances (EPS), which can be composed of proteins, polysaccharides, lipids and DNA are a major component of these mats and may function in the redistribution of nutrients and metabolites within the community. To test this notion, we established a model mat-building culture for comparison with the phylogenetically diverse natural mat communities. In these two systems we determined how proteins and glycans in the matrix changed as a function of light and tracked nutrient flow from the matrix. Using mass spectrometry metaproteomics analysis, we found homologous proteins in both field and culture extracellular matrix that point to cyanobacterial turnover of amino acids, inorganic nutrients, carbohydrates and nucleic acids from the EPS. Other abundant functions identified included oxidative stress response from both the cyanobacteria and heterotrophs and cyanobacterial structural proteins that may play a role in mat cohesion. Several degradative enzymes also varied in abundance in the EPS in response to light availability, suggesting active secretion. To further test cyanobacterial EPS turnover, we generated isotopically-labeled EPS and used NanoSIMS to trace uptake of this labeled EPS. Our findings suggest Cyanobacteria may facilitate nutrient transfer to other groups, as well as uptake of their own products through degradation of EPS components. This work provides evidence for the essential roles of EPS for storage, structural

  3. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

    PubMed

    Watson, Steve P

    2009-01-01

    The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

  4. Extracellular Matrix Properties Regulate the Migratory Response of Glioblastoma Stem Cells in Three-Dimensional Culture

    PubMed Central

    Herrera-Perez, Marisol; Voytik-Harbin, Sherry L.

    2015-01-01

    Diffuse infiltration across brain tissue is a hallmark of glioblastoma and the main cause of unsuccessful total resection that leads to tumor reappearance. A subpopulation termed glioblastoma stem cells (GSCs) has been directly related to aggressive invasion; nonetheless, their migratory characteristics and regulation by the microenvironment are still unknown. In this study, we developed a composite matrix of hyaluronan (HA) structurally supported by a collagen-oligomer fibril network to simulate the brain tumor extracellular matrix (ECM) composition. Matrigel-coated microfibers were embedded within the matrix to create a tunable dual niche microenvironment that resembles the vascular network of the brain. This model was compared with the most commonly used in vitro three-dimensional (3D) culture formats, Matrigel and collagen type-I monomer matrices, to study how the mechanical and compositional properties of the ECM alter the migration characteristics of GSC neurospheres. The migration mode, distance, velocity, and morphology of the GSCs were monitored over a 72-h period. The cells altered their migration mode depending on the matrix composition, showing migration by expansive growth in Matrigel matrices, multicellular extension along rigid interfaces (as Matrigel glass and coated microfibers), and mesenchymal single-cell migration in collagen matrices. Velocity and distance of migration within each composition varied according to matrix mechanical properties. In the dual niche system, the presence of HA reduced velocity and number of migratory cells; however, cells that came in contact with the pseudovessels exhibited collective migration by an extensive strand and reached higher velocities than cells migrating individually across the 3D matrix. Our results show that GSCs adopt varied migration mechanisms to invade multiple ECM microenvironments, and the migration characteristics exhibited are highly influenced by the matrix physical properties. Moreover, GSC

  5. [Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

    PubMed

    Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

    2013-01-01

    After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

  6. Extracellular Matrix Modulation: Optimizing Skin Care and Rejuvenation Procedures.

    PubMed

    Widgerow, Alan D; Fabi, Sabrina G; Palestine, Roberta F; Rivkin, Alexander; Ortiz, Arisa; Bucay, Vivian W; Chiu, Annie; Naga, Lina; Emer, Jason; Chasan, Paul E

    2016-04-01

    Normal aging and photoaging of the skin are chronic processes that progress gradually. The extracellular matrix (ECM), constituting over 70% of the skin, is the central hub for repair and regeneration of the skin. As such, the ECM is the area where changes related to photodamage are most evident. Degradation of the ECM with fragmentation of proteins significantly affects cross talk and signaling between cells, the matrix, and its constituents. The accumulation of collagen fragments, amorphous elastin agglutinations, and abnormal cross-linkages between the collagen fragments impedes the ECM from its normal repair and regenerative capacity, which manifests as wrinkled, non-elastic skin. Similar to how the chronic wound healing process requires wound bed preparation before therapeutic intervention, treatment of chronic aging of the skin would likely benefit from a "skin bed preparation" to optimize the outcome of rejuvenation procedures and skin maintenance programs. This involves introducing agents that can combat stress-induced oxidation, proteasome dysfunction, and non-enzymatic cross linkages involved in glycation end products, to collectively modulate this damaged ECM, and upregulate neocollagenesis and elastin production. Agents of particular interest are matrikines, peptides originating from the fragmentation of matrix proteins that exhibit a wide range of biological activities. Peptides of this type (tripeptide and hexapeptide) are incorporated in ALASTIN™ Skin Nectar with TriHex™ technology (ALASTIN Skincare, Inc., Carlsbad, CA), which is designed to target ECM modulation with a goal of optimizing results following invasive and non-invasive dermal rejuvenating procedures.

  7. Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings

    PubMed Central

    2012-01-01

    Background The design of biomimetic materials that parallel the morphology and biology of extracellular matrixes is key to the ability to grow functional tissues in vitro and to enhance the integration of biomaterial implants into existing tissues in vivo. Special attention has been put into mimicking the nanostructures of the extracellular matrix of bone, as there is a need to find biomaterials that can enhance the bonding between orthopedic devices and this tissue. Methods We have tested the ability of normal human osteoblasts to propagate and differentiate on silicon dioxide nanosprings, which can be easily grown on practically any surface. In addition, we tested different metals and metal alloys as coats for the nanosprings in tissue culture experiments with bone cells. Results Normal human osteoblasts grown on coated nanosprings exhibited an enhanced rate of propagation, differentiation into bone forming cells and mineralization. While osteoblasts did not attach effectively to bare nanowires grown on glass, these cells propagated successfully on nanosprings coated with titanium oxide and gold. We observed a 270 fold increase in the division rate of osteoblasts when grow on titanium/gold coated nanosprings. This effect was shown to be dependent on the nanosprings, as the coating by themselves did not alter the growth rate of osteoblast. We also observed that titanium/zinc/gold coated nanosprings increased the levels of osteoblast production of alkaline phosphatase seven folds. This result indicates that osteoblasts grown on this metal alloy coated nanosprings are differentiating to mature bone making cells. Consistent with this hypothesis, we showed that osteoblasts grown on the same metal alloy coated nanosprings have an enhanced ability to deposit calcium salt. Conclusion We have established that metal/metal alloy coated silicon dioxide nanosprings can be used as a biomimetic material paralleling the morphology and biology of osteogenic extracellular matrix

  8. Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings.

    PubMed

    Hass, Jamie L; Garrison, Erin M; Wicher, Sarah A; Knapp, Ben; Bridges, Nathan; McLlroy, Dn; Arrizabalaga, Gustavo

    2012-01-27

    The design of biomimetic materials that parallel the morphology and biology of extracellular matrixes is key to the ability to grow functional tissues in vitro and to enhance the integration of biomaterial implants into existing tissues in vivo. Special attention has been put into mimicking the nanostructures of the extracellular matrix of bone, as there is a need to find biomaterials that can enhance the bonding between orthopedic devices and this tissue. We have tested the ability of normal human osteoblasts to propagate and differentiate on silicon dioxide nanosprings, which can be easily grown on practically any surface. In addition, we tested different metals and metal alloys as coats for the nanosprings in tissue culture experiments with bone cells. Normal human osteoblasts grown on coated nanosprings exhibited an enhanced rate of propagation, differentiation into bone forming cells and mineralization. While osteoblasts did not attach effectively to bare nanowires grown on glass, these cells propagated successfully on nanosprings coated with titanium oxide and gold. We observed a 270 fold increase in the division rate of osteoblasts when grow on titanium/gold coated nanosprings. This effect was shown to be dependent on the nanosprings, as the coating by themselves did not alter the growth rate of osteoblast. We also observed that titanium/zinc/gold coated nanosprings increased the levels of osteoblast production of alkaline phosphatase seven folds. This result indicates that osteoblasts grown on this metal alloy coated nanosprings are differentiating to mature bone making cells. Consistent with this hypothesis, we showed that osteoblasts grown on the same metal alloy coated nanosprings have an enhanced ability to deposit calcium salt. We have established that metal/metal alloy coated silicon dioxide nanosprings can be used as a biomimetic material paralleling the morphology and biology of osteogenic extracellular matrix. The coated nanosprings enhance normal

  9. Adaptive rheology and ordering of cell cytoskeleton govern matrix rigidity sensing

    PubMed Central

    Gupta, Mukund; Sarangi, Bibhu Ranjan; Deschamps, Joran; Nematbakhsh, Yasaman; Callan-Jones, Andrew; Margadant, Felix; Mège, René-Marc; Lim, Chwee Teck; Voituriez, Raphaël; Ladoux, Benoît

    2015-01-01

    Matrix rigidity sensing regulates a large variety of cellular processes and has important implications for tissue development and disease. However, how cells probe matrix rigidity, and hence respond to it, remains unclear. Here, we show that rigidity sensing and adaptation emerge naturally from actin cytoskeleton remodeling. Our in vitro experiments and theoretical modeling demonstrate a bi-phasic rheology of the actin cytoskeleton, which transitions from fluid on soft substrates to solid on stiffer ones. Furthermore, we find that increasing substrate stiffness correlates with the emergence of an orientational order in actin stress fibers, which exhibit an isotropic to nematic transition that we characterize quantitatively in the framework of active matter theory. These findings imply mechanisms mediated by a large-scale reinforcement of actin structures under stress, which could be the mechanical drivers of substrate stiffness dependent cell shape changes and cell polarity. PMID:26109233

  10. Hypoxia and the extracellular matrix: drivers of tumour metastasis

    PubMed Central

    Gilkes, Daniele M.; Semenza, Gregg L.; Wirtz, Denis

    2014-01-01

    Of the deaths attributed to cancer, 90% are due to metastasis, and treatments that prevent or cure metastasis remain elusive. Emerging data indicate that hypoxia and the extracellular matrix (ECM) might have crucial roles in metastasis. During tumour evolution, changes in the composition and the overall content of the ECM reflect both its biophysical and biological properties and these strongly influence tumour and stromal cell properties, such as proliferation and motility. Originally thought of as independent contributors to metastatic spread, recent studies have established a direct link between hypoxia and the composition and the organization of the ECM, which suggests a new model in which multiple microenvironmental signals might converge to synergistically influence metastatic outcome. PMID:24827502

  11. Extracellular Matrix and Fibroblast Communication Following Myocardial Infarction

    PubMed Central

    Ma, Yonggang; Halade, Ganesh V.; Lindsey, Merry L.

    2012-01-01

    The extracellular matrix (ECM) provides structural support by serving as a scaffold for cells, and as such the ECM maintains normal tissue homeostasis and mediates the repair response following injury. In response to myocardial infarction (MI), ECM expression is generally upregulated in the left ventricle (LV), which regulates LV remodeling by modulating scar formation. The ECM directly affects scar formation by regulating growth factor release and cell adhesion, and indirectly affects scar formation by regulating the inflammatory, angiogenic, and fibroblast responses. This review summarizes the current literature on ECM expression patterns and fibroblast mechanisms in the myocardium, focusing on the ECM response to MI. In addition, we discuss future research areas that are needed to better understand the molecular mechanisms of ECM action, both in general and as a means to optimize infarct healing. PMID:22926488

  12. Extracellular matrix hydrogels from decellularized tissues: Structure and function.

    PubMed

    Saldin, Lindsey T; Cramer, Madeline C; Velankar, Sachin S; White, Lisa J; Badylak, Stephen F

    2017-02-01

    Extracellular matrix (ECM) bioscaffolds prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications. The discovery that these ECM materials could be solubilized and subsequently manipulated to form hydrogels expanded their potential in vitro and in vivo utility; i.e. as culture substrates comparable to collagen or Matrigel, and as injectable materials that fill irregularly-shaped defects. The mechanisms by which ECM hydrogels direct cell behavior and influence remodeling outcomes are only partially understood, but likely include structural and biological signals retained from the native source tissue. The present review describes the utility, formation, and physical and biological characterization of ECM hydrogels. Two examples of clinical application are presented to demonstrate in vivo utility of ECM hydrogels in different organ systems. Finally, new research directions and clinical translation of ECM hydrogels are discussed.

  13. Extracellular matrix production in vitro in cartilage tissue engineering.

    PubMed

    Chen, Jie-Lin; Duan, Li; Zhu, Weimin; Xiong, Jianyi; Wang, Daping

    2014-04-05

    Cartilage tissue engineering is arising as a technique for the repair of cartilage lesions in clinical applications. However, fibrocartilage formation weakened the mechanical functions of the articular, which compromises the clinical outcomes. Due to the low proliferation ability, dedifferentiation property and low production of cartilage-specific extracellular matrix (ECM) of the chondrocytes, the cartilage synthesis in vitro has been one of the major limitations for obtaining high-quality engineered cartilage constructs. This review discusses cells, biomaterial scaffolds and stimulating factors that can facilitate the cartilage-specific ECM production and accumulation in the in vitro culture system. Special emphasis has been put on the factors that affect the production of ECM macromolecules such as collagen type II and proteoglycans in the review, aiming at providing new strategies to improve the quality of tissue-engineered cartilage.

  14. Insight On Colorectal Carcinoma Infiltration by Studying Perilesional Extracellular Matrix.

    PubMed

    Nebuloni, Manuela; Albarello, Luca; Andolfo, Annapaola; Magagnotti, Cinzia; Genovese, Luca; Locatelli, Irene; Tonon, Giovanni; Longhi, Erika; Zerbi, Pietro; Allevi, Raffaele; Podestà, Alessandro; Puricelli, Luca; Milani, Paolo; Soldarini, Armando; Salonia, Andrea; Alfano, Massimo

    2016-03-04

    The extracellular matrix (ECM) from perilesional and colorectal carcinoma (CRC), but not healthy colon, sustains proliferation and invasion of tumor cells. We investigated the biochemical and physical diversity of ECM in pair-wised comparisons of healthy, perilesional and CRC specimens. Progressive linearization and degree of organization of fibrils was observed from healthy to perilesional and CRC ECM, and was associated with a steady increase of stiffness and collagen crosslinking. In the perilesional ECM these modifications coincided with increased vascularization, whereas in the neoplastic ECM they were associated with altered modulation of matrisome proteins, increased content of hydroxylated lysine and lysyl oxidase. This study identifies the increased stiffness and crosslinking of the perilesional ECM predisposing an environment suitable for CRC invasion as a phenomenon associated with vascularization. The increased stiffness of colon areas may represent a new predictive marker of desmoplastic region predisposing to invasion, thus offering new potential application for monitoring adenoma with invasive potential.

  15. Insight On Colorectal Carcinoma Infiltration by Studying Perilesional Extracellular Matrix

    PubMed Central

    Nebuloni, Manuela; Albarello, Luca; Andolfo, Annapaola; Magagnotti, Cinzia; Genovese, Luca; Locatelli, Irene; Tonon, Giovanni; Longhi, Erika; Zerbi, Pietro; Allevi, Raffaele; Podestà, Alessandro; Puricelli, Luca; Milani, Paolo; Soldarini, Armando; Salonia, Andrea; Alfano, Massimo

    2016-01-01

    The extracellular matrix (ECM) from perilesional and colorectal carcinoma (CRC), but not healthy colon, sustains proliferation and invasion of tumor cells. We investigated the biochemical and physical diversity of ECM in pair-wised comparisons of healthy, perilesional and CRC specimens. Progressive linearization and degree of organization of fibrils was observed from healthy to perilesional and CRC ECM, and was associated with a steady increase of stiffness and collagen crosslinking. In the perilesional ECM these modifications coincided with increased vascularization, whereas in the neoplastic ECM they were associated with altered modulation of matrisome proteins, increased content of hydroxylated lysine and lysyl oxidase. This study identifies the increased stiffness and crosslinking of the perilesional ECM predisposing an environment suitable for CRC invasion as a phenomenon associated with vascularization. The increased stiffness of colon areas may represent a new predictive marker of desmoplastic region predisposing to invasion, thus offering new potential application for monitoring adenoma with invasive potential. PMID:26940881

  16. The extracellular matrix: A dynamic niche in cancer progression

    PubMed Central

    Lu, Pengfei; Weaver, Valerie M.

    2012-01-01

    The local microenvironment, or niche, of a cancer cell plays important roles in cancer development. A major component of the niche is the extracellular matrix (ECM), a complex network of macromolecules with distinctive physical, biochemical, and biomechanical properties. Although tightly controlled during embryonic development and organ homeostasis, the ECM is commonly deregulated and becomes disorganized in diseases such as cancer. Abnormal ECM affects cancer progression by directly promoting cellular transformation and metastasis. Importantly, however, ECM anomalies also deregulate behavior of stromal cells, facilitate tumor-associated angiogenesis and inflammation, and thus lead to generation of a tumorigenic microenvironment. Understanding how ECM composition and topography are maintained and how their deregulation influences cancer progression may help develop new therapeutic interventions by targeting the tumor niche. PMID:22351925

  17. Hydrogels as extracellular matrix mimics for 3D cell culture.

    PubMed

    Tibbitt, Mark W; Anseth, Kristi S

    2009-07-01

    Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two-dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three-dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three-dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three-dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic-biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism. (c) 2009 Wiley Periodicals, Inc.

  18. Extracellular matrix bioscaffolds in tissue remodeling and morphogenesis

    PubMed Central

    Swinehart, Ilea T.; Badylak, Stephen F.

    2016-01-01

    During normal morphogenesis the extracellular matrix (ECM) influences cell motility, proliferation, apoptosis, and differentiation. Tissue engineers have attempted to harness the cell signaling potential of ECM to promote the functional reconstruction, if not regeneration, of injured or missing adult tissues that otherwise heal by the formation of scar tissue. ECM bioscaffolds, derived from decellularized tissues, have been used to promote the formation of site appropriate, functional tissues in many clinical applications including skeletal muscle, fibrocartilage, lower urinary tract, and esophageal reconstruction, among others. These scaffolds function by the release or exposure of growth factors and cryptic peptides, modulation of the immune response, and recruitment of progenitor cells. Herein, we describe this process of ECM induced constructive remodeling and examine similarities to normal tissue morphogenesis. PMID:26699796

  19. Extracellular Matrix and Integrins in Embryonic Stem Cell Differentiation

    PubMed Central

    Wang, Han; Luo, Xie; Leighton, Jake

    2015-01-01

    Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM–integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study. PMID:26462244

  20. Domain structure and organisation in extracellular matrix proteins.

    PubMed

    Hohenester, Erhard; Engel, Jürgen

    2002-03-01

    Extracellular matrix (ECM) proteins are large modular molecules built up from a limited set of modules, or domains. The basic folds of many domains have now been determined by crystallography or NMR spectroscopy. Recent structures of domain pairs and larger tandem arrays, as well as of oligomerisation domains, have begun to reveal the principles underlying the higher order architecture of ECM proteins. Structural information, coupled with site-directed mutagenesis, has been instrumental in showing how adjacent domains can co-operate in ligand binding. Very recently, the first heterotypic ECM protein complexes have become available. Here, we review the advances of the last 5 years in understanding ECM protein structure, with special emphasis on those structures that have given insight into the biological functions of ECM proteins.

  1. Constructive remodeling of a synthetic endothelial extracellular matrix

    PubMed Central

    Han, Sewoon; Shin, Yoojin; Jeong, Hyo Eun; Jeon, Jessie S.; Kamm, Roger D.; Huh, Dongeun; Sohn, Lydia L.; Chung, Seok

    2015-01-01

    The construction of well-controllable in vitro models of physiological and pathological vascular endothelium remains a fundamental challenge in tissue engineering and drug development. Here, we present an approach for forming a synthetic endothelial extracellular matrix (ECM) that closely resembles that of the native structure by locally depositing basement membrane materials onto type 1 collagen nanofibers only in a region adjacent to the endothelial cell (EC) monolayer. Culturing the EC monolayer on this synthetic endothelial ECM remarkably enhanced its physiological properties, reducing its vascular permeability, and promoting a stabilized, quiescent phenotype. We demonstrated that the EC monolayer on the synthetic endothelial ECM neither creates non-physiological barriers to cell-cell or cell-ECM interactions, nor hinders molecular diffusion of growth factors and other molecules. The synthetic endothelial ECM and vascular endothelium on it may help us enter in a new phase of research in which various models of the biological barrier behavior can be tested experimentally. PMID:26687334

  2. Preparation of Cardiac Extracellular Matrix from an Intact Porcine Heart

    PubMed Central

    Wainwright, John M.; Czajka, Caitlin A.; Patel, Urvi B.; Freytes, Donald O.; Tobita, Kimimasa; Gilbert, Thomas W.

    2010-01-01

    Whole organ engineering would benefit from a three-dimensional scaffold produced from intact organ-specific extracellular matrix (ECM). The microenvironment and architecture provided by such a scaffold would likely support site-appropriate cell differentiation and spatial organization. The methods to produce such scaffolds from intact organs require customized decellularization protocols. In the present study, intact adult porcine hearts were successfully decellularized in less than 10 h using pulsatile retrograde aortic perfusion. Serial perfusion of an enzymatic, nonionic detergent, ionic detergent, and acid solution with hypotonic and hypertonic rinses was used to systematically remove cellular content. The resultant cardiac ECM retained collagen, elastin, and glycosaminoglycans, and mechanical integrity. Cardiac ECM supported the formation of organized chicken cardiomyocyte sarcomere structure in vitro. The intact decellularized porcine heart provides a tissue engineering template that may be beneficial for future preclinical studies and eventual clinical applications. PMID:19702513

  3. Preparation of cardiac extracellular matrix from an intact porcine heart.

    PubMed

    Wainwright, John M; Czajka, Caitlin A; Patel, Urvi B; Freytes, Donald O; Tobita, Kimimasa; Gilbert, Thomas W; Badylak, Stephen F

    2010-06-01

    Whole organ engineering would benefit from a three-dimensional scaffold produced from intact organ-specific extracellular matrix (ECM). The microenvironment and architecture provided by such a scaffold would likely support site-appropriate cell differentiation and spatial organization. The methods to produce such scaffolds from intact organs require customized decellularization protocols. In the present study, intact adult porcine hearts were successfully decellularized in less than 10 h using pulsatile retrograde aortic perfusion. Serial perfusion of an enzymatic, nonionic detergent, ionic detergent, and acid solution with hypotonic and hypertonic rinses was used to systematically remove cellular content. The resultant cardiac ECM retained collagen, elastin, and glycosaminoglycans, and mechanical integrity. Cardiac ECM supported the formation of organized chicken cardiomyocyte sarcomere structure in vitro. The intact decellularized porcine heart provides a tissue engineering template that may be beneficial for future preclinical studies and eventual clinical applications.

  4. Decellularization of Rat Kidneys to Produce Extracellular Matrix Scaffolds.

    PubMed

    Jin, Mei; Yaling, Yu; Zhibin, Wang; Jianse, Zhang

    2016-01-01

    The extracellular matrix (ECM) retains three-dimensional structures for the stimulation of cell growth, with components of the ECM relatively conserved between species. Interest in the use of decellularized scaffold-based strategies for organ regeneration is increasing rapidly. Decellularized scaffolds derived from animal organs are a promising material for organ engineering, with a number of prominent advances having been reported in the past few years.In this article we describe a simple and robust methodology for generating decellularized rat kidneys. To obtain these scaffolds, we perfuse rat kidneys with detergents through the abdominal aorta. After decellularization, kidney scaffolds are harvested for evaluation of vascular structure and histology. Qualitative evaluation involves vascular corrosion casting, transmission electron microscopy, and several different histological and immunofluorescent methods. SDS residue levels are assessed by ultraviolet-visible spectrophotometer (UV-VIS).

  5. Aging-associated changes in renal extracellular matrix.

    PubMed Central

    Abrass, C. K.; Adcox, M. J.; Raugi, G. J.

    1995-01-01

    The composition of renal extracellular matrices was examined in 6-, 12-, 18-, and 24-month-old rats by immunofluorescence microscopy. No change in composition of tubular basement membrane was detected. Increased immunostaining for laminin chains B1 and s-laminin and thrombospondin characterized the thickened glomerular basement membrane. Interstitial collagens I and III were not detected in globally sclerotic glomeruli. The major change noted in the aged rat kidney at 24 months was generalized expansion of the interstitium by thrombospondin and fibronectin. In areas of tubular atrophy there was new expression of extra domain A (EDA)+ fibronectin. Collagens I and III were detected focally in the interstitium adjacent to areas of tubular atrophy, but otherwise collagens I, III, and IV and laminin did not contribute to the interstitial fibrosis. Interstitial fibrosis was detectable at 18 months of age and preceded the development of sclerotic glomeruli, tubular atrophy, or accumulations of interstitial collagen. These changes in extracellular matrix composition distinguish the aging kidney from other sclerotic forms of renal disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7887455

  6. Cells involved in extracellular matrix remodeling after acute myocardial infarction

    PubMed Central

    Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

    2015-01-01

    Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. PMID:25993074

  7. Role of Extracellular Matrix-Mediated Interactions in Thymocyte Migration

    PubMed Central

    Dalmau, Sérgio Ranto; Dealmeida, Vinícius Cotta

    2000-01-01

    Cell adhesion, migration, differentiation and survival or death is amongst a large spectrum of biological responses that can be elicited by ligation of extracellular matrix components to their corresponding receptors. As regards the physiology of the thymus, cell migration is a crucial event in the general process of T cell differentiation. Studies on the intrathymic distribution of ECM components revealed that fibronectin, laminin and type IV collagen, are not restrictedly located at typical basement membrane sites, also forming a thick network in the medullary region of the thymic lobules, whereas very thin ECM fibers are found within the cortex. These ECM components are essentially produced by thymic microenvironmental cells, which also drive thymocyte differentiation. Signals triggered by ECM are conveyed into thymocytes or microenvironmental cells through specific membrane receptors, and most of them belong to the integrin type, such as the VLA-3, VLA-4, VLA-5 and VLA-6. In vitro studies revealed that adhesion of thymocytes to thymic microenvironmental cells is mediated by extracellular matrix. Such an adhesion is preferentially done by immature thymocytes. Importantly, ECM-mediated interactions also govern the entrance and exit of thymocytes in the lymphoepithelial complexes named thymic nurse cells. Lastly, pathological conditions, including infectious and autoimmune diseases, in which changes of ECM ligands and receptors are observed, course with alterations in thymocyte migration and death. In conclusion, the fact that ECM can modulate traffic, differentiation, death and survival of normal thymocytes adds clues for understanding how ECM-mediated interactions behave in the thymus, not only in normal, but also in pathological conditions. PMID:11097218

  8. Extracellular matrix assembly and organization during zebrafish gastrulation.

    PubMed

    Latimer, Andrew; Jessen, Jason R

    2010-03-01

    Zebrafish gastrulation entails morphogenetic cell movements that shape the body plan and give rise to an embryo with defined anterior-posterior and dorsal-ventral axes. Regulating these cell movements are diverse signaling pathways and proteins including Wnts, Src-family tyrosine kinases, cadherins, and matrix metalloproteinases. While our knowledge of how these proteins impact cell polarity and migration has advanced considerably in the last decade, almost no data exist regarding the organization of extracellular matrix (ECM) during zebrafish gastrulation. Here, we describe for the first time the assembly of a fibronectin (FN) and laminin containing ECM in the early zebrafish embryo. This matrix was first detected at early gastrulation (65% epiboly) in the form of punctae that localize to tissue boundaries separating germ layers from each other and the underlying yolk cell. Fibrillogenesis increased after mid-gastrulation (80% epiboly) coinciding with the period of planar cell polarity pathway-dependent convergence and extension cell movements. We demonstrate that FN fibrils present beneath deep mesodermal cells are aligned in the direction of membrane protrusion formation. Utilizing antisense morpholino oligonucleotides, we further show that knockdown of FN expression causes a convergence and extension defect. Taken together, our data show that similar to amphibian embryos, the formation of ECM in the zebrafish gastrula is a dynamic process that occurs in parallel to at least a portion of the polarized cell behaviors shaping the embryonic body plan. These results provide a framework for uncovering the interrelationship between ECM structure and cellular processes regulating convergence and extension such as directed migration and mediolateral/radial intercalation. 2009 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  9. Targeting extracellular matrix remodeling in disease: Could resveratrol be a potential candidate?

    PubMed

    Agarwal, Renu; Agarwal, Puneet

    2017-02-01

    Disturbances of extracellular matrix homeostasis are associated with a number of pathological conditions. The ability of extracellular matrix to provide contextual information and hence control the individual or collective cellular behavior is increasingly being recognized. Hence, newer therapeutic approaches targeting extracellular matrix remodeling are widely investigated. We reviewed the current literature showing the effects of resveratrol on various aspects of extracellular matrix remodeling. This review presents a summary of the effects of resveratrol on extracellular matrix deposition and breakdown. Mechanisms of action of resveratrol in extracellular matrix deposition involving growth factors and their signaling pathways are discussed. Involvement of phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways and role of transcription factors and sirtuins on the effects of resveratrol on extracellular matrix homeostasis are summarized. It is evident from the literature presented in this review that resveratrol has significant effects on both the synthesis and breakdown of extracellular matrix. The major molecular targets of the action of resveratrol are growth factors and their signaling pathways, phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways, transcription factors, and SIRT-1. The effects of resveratrol on extracellular matrix and the molecular targets appear to be related to experimental models, experimental environment as well as the doses.

  10. Tailoring material properties of a nanofibrous extracellular matrix derived hydrogel

    PubMed Central

    Johnson, Todd D.; Lin, Stephen Y.; Christman, Karen L.

    2012-01-01

    In the native tissue, the interaction between cells and the extracellular matrix (ECM) is essential for cell migration, proliferation, differentiation, mechanical stability, and signaling. It has been shown that decellularized ECMs can be processed into injectable formulations, thereby allowing for minimally invasive delivery. Upon injection and increase in temperature, these materials self-assemble into porous gels forming a complex network of fibers with nano-scale structure. In this study we aimed to examine and tailor the material properties of a self-assembling ECM hydrogel derived from porcine myocardial tissue, which was developed as a tissue specific injectable scaffold for cardiac tissue engineering. The impact of gelation parameters on ECM hydrogels has not previously been explored. We examined how modulating pH, temperature, ionic strength, and concentration affected the nanoscale architecture, mechanical properties, and gelation kinetics. These material characteristics were assessed using scanning electron microscopy, rheometry, and spectrophotometry, respectively. Since the main component of the myocardial matrix is collagen, many similarities between the ECM hydrogel and collagen gels were observed in terms of the nanofibrous structure and modulation of properties by altering ionic strength. However, variation from collagen gels was noted for the gelation temperature along with varied times and rates of gelation. These discrepancies when compared to collagen are likely due to the presence of other ECM components in the decellularized ECM based hydrogel. These results demonstrate how the material properties of ECM hydrogels could be tailored for future in vitro and in vivo applications. PMID:22101810

  11. Extracellular matrix: A dynamic microenvironment for stem cell niche☆

    PubMed Central

    Gattazzo, Francesca; Urciuolo, Anna; Bonaldo, Paolo

    2014-01-01

    Background Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue and able to regulate cell behavior. Stem cells have a key role in the maintenance and regeneration of tissues and they are located in a specific microenvironment, defined as niche. Scope of review We overview the progresses that have been made in elucidating stem cell niches and discuss the mechanisms by which ECM affects stem cell behavior. We also summarize the current tools and experimental models for studying ECM–stem cell interactions. Major conclusions ECM represents an essential player in stem cell niche, since it can directly or indirectly modulate the maintenance, proliferation, self-renewal and differentiation of stem cells. Several ECM molecules play regulatory functions for different types of stem cells, and based on its molecular composition the ECM can be deposited and finely tuned for providing the most appropriate niche for stem cells in the various tissues. Engineered biomaterials able to mimic the in vivo characteristics of stem cell niche provide suitable in vitro tools for dissecting the different roles exerted by the ECM and its molecular components on stem cell behavior. General significance ECM is a key component of stem cell niches and is involved in various aspects of stem cell behavior, thus having a major impact on tissue homeostasis and regeneration under physiological and pathological conditions. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. PMID:24418517

  12. Domain organizations of modular extracellular matrix proteins and their evolution.

    PubMed

    Engel, J

    1996-11-01

    Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

  13. Tailoring material properties of a nanofibrous extracellular matrix derived hydrogel

    NASA Astrophysics Data System (ADS)

    Johnson, Todd D.; Lin, Stephen Y.; Christman, Karen L.

    2011-12-01

    In the native tissue, the interaction between cells and the extracellular matrix (ECM) is essential for cell migration, proliferation, differentiation, mechanical stability, and signaling. It has been shown that decellularized ECMs can be processed into injectable formulations, thereby allowing for minimally invasive delivery. Upon injection and increase in temperature, these materials self-assemble into porous gels forming a complex network of fibers with nanoscale structure. In this study we aimed to examine and tailor the material properties of a self-assembling ECM hydrogel derived from porcine myocardial tissue, which was developed as a tissue specific injectable scaffold for cardiac tissue engineering. The impact of gelation parameters on ECM hydrogels has not previously been explored. We examined how modulating pH, temperature, ionic strength, and concentration affected the nanoscale architecture, mechanical properties, and gelation kinetics. These material characteristics were assessed using scanning electron microscopy, rheometry, and spectrophotometry, respectively. Since the main component of the myocardial matrix is collagen, many similarities between the ECM hydrogel and collagen gels were observed in terms of the nanofibrous structure and modulation of properties by altering ionic strength. However, variation from collagen gels was noted for the gelation temperature along with varied times and rates of gelation. These discrepancies when compared to collagen are likely due to the presence of other ECM components in the decellularized ECM based hydrogel. These results demonstrate how the material properties of ECM hydrogels could be tailored for future in vitro and in vivo applications.

  14. Matrix rigidity differentially regulates invadopodia activity through ROCK1 and ROCK2.

    PubMed

    Jerrell, Rachel J; Parekh, Aron

    2016-04-01

    ROCK activity increases due to ECM rigidity in the tumor microenvironment and promotes a malignant phenotype via actomyosin contractility. Invasive migration is facilitated by actin-rich adhesive protrusions known as invadopodia that degrade the ECM. Invadopodia activity is dependent on matrix rigidity and contractile forces suggesting that mechanical factors may regulate these subcellular structures through ROCK-dependent actomyosin contractility. However, emerging evidence indicates that the ROCK1 and ROCK2 isoforms perform different functions in cells suggesting that alternative mechanisms may potentially regulate rigidity-dependent invadopodia activity. In this study, we found that matrix rigidity drives ROCK signaling in cancer cells but that ROCK1 and ROCK2 differentially regulate invadopodia activity through separate signaling pathways via contractile (NM II) and non-contractile (LIMK) mechanisms. These data suggest that the mechanical rigidity of the tumor microenvironment may drive ROCK signaling through distinct pathways to enhance the invasive migration required for cancer progression and metastasis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture.

    PubMed

    Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-Ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

    2010-01-15

    Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.

  16. Extracellular Matrix Induced Gene Expression in Human Breast Cancer Cells

    PubMed Central

    Garamszegi, Nandor; Garamszegi, Susanna P.; Shehadeh, Lina A.; Scully, Sean P.

    2009-01-01

    Extracellular matrix (ECM) molecules modify gene expression through attachment-dependent (i.e., focal adhesion related) integrin receptor signalling. It was previously unknown whether the same molecules acting as soluble peptides could generate signal cascades without the associated mechanical anchoring, a condition that may be encountered during matrix remodelling, degradation and relevant to invasion and metastatic processes. In the current study the role of ECM ligand regulated gene expression through this attachment independent process was examined. It was observed that fibronectin, laminin, collagens type I and II induce Smad2 activation in MCF-10A and MCF-7 cells. This activation is not caused by TGFβ ligand contamination or autocrine TGF involvement and is 3–5 fold less robust than the TGFβ1 ligand. The resulting nuclear translocation of Smad4 in response to ECM ligand indicates downstream transcriptional responses occurring. Co-immunoprecipitation experiments determined that type II collagen and laminin act through interaction with integrin α2β1 receptor complex. The ECM ligand induced Smad activation (termed signalling crosstalk) resulted cell type and ligand specific transcriptional changes which are distinct from the TGFβ ligand induced responses. These findings demonstrate that cell-matrix communication is more complex than previously thought. Soluble ECM peptides drive transcriptional regulation through corresponding adhesion and non-attachment related processes. The resultant gene expressional patterns correlate with pathway activity and not by the extent of Smad activation. These results extend the complexity and the existing paradigms of ECM-cell communication to ECM ligand regulation without the necessity of mechanical coupling. PMID:19276183

  17. [Single mechanism of remodelling extracellular matrix in thymus and pineal gland at aging].

    PubMed

    Lin'kova, N S; Poliakova, V O; Kvetnoĭ, I M

    2011-01-01

    The expression of matrix metalloproteinase 2 and 9 in thymus and pineal gland has been verified. These data demonstrate single mechanism of remodelling extracellular matrix in thymus and pineal gland at aging.

  18. Pseudomonas aeruginosa binds to extracellular matrix deposited by human corneal epithelial cells.

    PubMed

    Esco, Miechia A; Hazlett, Linda D; Kurpakus-Wheater, Michelle

    2002-12-01

    To measure the effect of extracellular matrix substrate, pH, and O(2) on Pseudomonas aeruginosa binding. Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O(2). P. aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O(2). The mean number of adherent bacteria per counted per field +/- SEM (n = 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O(2). Binding in the presence of antibodies directed against laminin-5 was also measured. Extracellular matrix substrates produced by cells cultured in 20% O(2), combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660. In contrast, extracellular matrix substrates produced by cells cultured in 2% O(2), combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660. Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660. Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control. The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P. aeruginosa. Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix. The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion.

  19. Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development

    PubMed Central

    Stanic, Karen; Saldivia, Natalia; Förstera, Benjamín; Torrejón, Marcela; Montecinos, Hernán; Caprile, Teresa

    2016-01-01

    Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons. PMID:27733818

  20. The tetrapartite synapse: extracellular matrix remodeling contributes to corticoaccumbens plasticity underlying drug addiction

    PubMed Central

    Smith, Alexander C.W.; Scofield, Michael D.; Kalivas, Peter W.

    2015-01-01

    Synaptic plasticity has long been known to involve three key elements of neuropil, the presynapse, the postsynapse and adjacent glia. Here we review the role of the extracellular matrix in synaptic plasticity as a necessary component forming the tetrapartite synapse. We describe the role of matrix metalloproteinases as enzymes sculpting extracellular proteins and thereby creating an extracellular signaling domain required for synaptic plasticity. Specifically we focus on the role of the tetrapartite synapse in mediating the effects of addictive drugs at corticostriatal synapses, and conclude that the extracellular signaling domain and its regulation by matrix metalloproteinases is critical for developing and expressing drug seeking behaviors. PMID:25838241

  1. Extracellular matrix inflammation in vascular cognitive impairment and dementia.

    PubMed

    Rosenberg, Gary A

    2017-03-01

    Vascular cognitive impairment and dementia (VCID) include a wide spectrum of chronic manifestations of vascular disease related to large vessel strokes and small vessel disease (SVD). Lacunar strokes and white matter (WM) injury are consequences of SVD. The main vascular risk factor for SVD is brain hypoperfusion from cerebral blood vessel narrowing due to chronic hypertension. The hypoperfusion leads to activation and degeneration of astrocytes with the resulting fibrosis of the extracellular matrix (ECM). Elasticity is lost in fibrotic cerebral vessels, reducing the response of stiffened blood vessels in times of increased metabolic need. Intermittent hypoxia/ischaemia activates a molecular injury cascade, producing an incomplete infarction that is most damaging to the deep WM, which is a watershed region for cerebral blood flow. Neuroinflammation caused by hypoxia activates microglia/macrophages to release proteases and free radicals that perpetuate the damage over time to molecules in the ECM and the neurovascular unit (NVU). Matrix metalloproteinases (MMPs) secreted in an attempt to remodel the blood vessel wall have the undesired consequences of opening the blood-brain barrier (BBB) and attacking myelinated fibres. This dual effect of the MMPs causes vasogenic oedema in WM and vascular demyelination, which are the hallmarks of the subcortical ischaemic vascular disease (SIVD), which is the SVD form of VCID also called Binswanger's disease (BD). Unravelling the complex pathophysiology of the WM injury-related inflammation in the small vessel form of VCID could lead to novel therapeutic strategies to reduce damage to the ECM, preventing the progressive damage to the WM. © 2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  2. Extracellular matrix protein expression is brain region dependent.

    PubMed

    Dauth, Stephanie; Grevesse, Thomas; Pantazopoulos, Harry; Campbell, Patrick H; Maoz, Ben M; Berretta, Sabina; Parker, Kevin Kit

    2016-05-01

    In the brain, extracellular matrix (ECM) components form networks that contribute to structural and functional diversity. Maladaptive remodeling of ECM networks has been reported in neurodegenerative and psychiatric disorders, suggesting that the brain microenvironment is a dynamic structure. A lack of quantitative information about ECM distribution in the brain hinders an understanding of region-specific ECM functions and the role of ECM in health and disease. We hypothesized that each ECM protein as well as specific ECM structures, such as perineuronal nets (PNNs) and interstitial matrix, are differentially distributed throughout the brain, contributing to the unique structure and function in the various regions of the brain. To test our hypothesis, we quantitatively analyzed the distribution, colocalization, and protein expression of aggrecan, brevican, and tenascin-R throughout the rat brain utilizing immunohistochemistry and mass spectrometry analysis and assessed the effect of aggrecan, brevican, and/or tenascin-R on neurite outgrowth in vitro. We focused on aggrecan, brevican, and tenascin-R as they are especially expressed in the mature brain, and have established roles in brain development, plasticity, and neurite outgrowth. The results revealed a differentiated distribution of all three proteins throughout the brain and indicated that their presence significantly reduces neurite outgrowth in a 3D in vitro environment. These results underline the importance of a unique and complex ECM distribution for brain physiology and suggest that encoding the distribution of distinct ECM proteins throughout the brain will aid in understanding their function in physiology and in turn assist in identifying their role in disease. J. Comp. Neurol. 524:1309-1336, 2016. © 2016 Wiley Periodicals, Inc.

  3. Astrocytes as a Source for Extracellular Matrix Molecules and Cytokines

    PubMed Central

    Wiese, Stefan; Karus, Michael; Faissner, Andreas

    2012-01-01

    Research of the past 25 years has shown that astrocytes do more than participating and building up the blood-brain barrier and detoxify the active synapse by reuptake of neurotransmitters and ions. Indeed, astrocytes express neurotransmitter receptors and, as a consequence, respond to stimuli. Within the tripartite synapse, the astrocytes owe more and more importance. Besides the functional aspects the differentiation of astrocytes has gained a more intensive focus. Deeper knowledge of the differentiation processes during development of the central nervous system might help explaining and even help treating neurological diseases like Alzheimer’s disease, Amyotrophic lateral sclerosis, Parkinsons disease, and psychiatric disorders in which astrocytes have been shown to play a role. Specific differentiation of neural stem cells toward the astroglial lineage is performed as a multi-step process. Astrocytes and oligodendrocytes develop from a multipotent stem cell that prior to this has produced primarily neuronal precursor cells. This switch toward the more astroglial differentiation is regulated by a change in receptor composition on the cell surface and responsiveness to Fibroblast growth factor and Epidermal growth factor (EGF). The glial precursor cell is driven into the astroglial direction by signaling molecules like Ciliary neurotrophic factor, Bone Morphogenetic Proteins, and EGF. However, the early astrocytes influence their environment not only by releasing and responding to diverse soluble factors but also express a wide range of extracellular matrix (ECM) molecules, in particular proteoglycans of the lectican family and tenascins. Lately these ECM molecules have been shown to participate in glial development. In this regard, especially the matrix protein Tenascin C (Tnc) proved to be an important regulator of astrocyte precursor cell proliferation and migration during spinal cord development. Nevertheless, ECM molecules expressed by reactive astrocytes

  4. The endogenous fluorescence of fibroblast in collagen gels as indicator of stiffness of the extracellular matrix

    NASA Astrophysics Data System (ADS)

    Padilla-Martinez, J. P.; Ortega-Martinez, A.; Franco, W.

    2016-03-01

    The stiffness or rigidity of the extracellular matrix (ECM) regulates cell response. Established mechanical tests to measure stiffness, such as indentation and tensile tests, are invasive and destructive to the sample. Endogenous or native molecules to cells and ECM components, like tryptophan and cross-links of collagen, display fluorescence upon irradiation with ultraviolet light. Most likely, the concentration of these endogenous fluorophores changes as the stiffness of the ECM changes. In this work we investigate the endogenous fluorescence of collagen gels containing fibroblasts as a non-invasive non-destructive method to measure stiffness of the ECM. Human fibroblast cells were cultured in three-dimensional gels of type I collagen (50,000 cells/ml). This construct is a simple model of tissue contraction. During contraction, changes in the excitation-emission matrix (a fluorescence map in the 240-520/290-530 nm range) of constructs were measured with a spectrofluoremeter, and changes in stiffness were measured with a standard indentation test over 16 days. Results show that a progressive increase in fluorescence of the 290/340 nm excitation-emission pair correlates with a progressive increase in stiffness (r=0.9, α=0.5). The fluorescence of this excitation-emission pair is ascribed to tryptophan and variations in the fluorescence of this pair correlate with cellular proliferation. In this tissue model, the endogenous functional fluorescence of proliferating fibroblast cells is a biomechanical marker of stiffness of the ECM.

  5. Force-induced elastic matrix-mediated interactions in the presence of a rigid wall

    NASA Astrophysics Data System (ADS)

    Menzel, Andreas M.

    We consider an elastic composite material containing particulate inclusions in a soft elastic matrix that is bounded by a rigid wall, e.g., the substrate. If such a composite serves as a soft actuator, forces are imposed on or induced between the embedded particles. We investigate how the presence of the rigid wall affects the interactions between the inclusions in the elastic matrix. For no-slip boundary conditions, we transfer Blake's derivation of a corresponding Green's function from low-Reynolds-number hydrodynamics to the linearly elastic case. Results for no-slip and free-slip surface conditions are compared to each other and to the bulk behavior. Our results suggest that walls with free-slip surface conditions are preferred when they serve as substrates for soft actuators made from elastic composite materials. As we further demonstrate, the presence of a rigid wall can qualitatively change the interactions between the inclusions. In effect, it can switch attractive interactions into repulsive ones (and vice versa). It should be straightforward to observe the effects in future experiments and to combine our results, e.g., with the modeling of biological cells and tissue on rigid surfaces.

  6. LRP1 regulates remodeling of the extracellular matrix by fibroblasts

    PubMed Central

    Gaultier, Alban; Hollister, Margaret; Reynolds, Irene; Hsieh, En-hui; Gonias, Steven L.

    2009-01-01

    Low density lipoprotein receptor-related protein (LRP1) is an endocytic receptor for diverse proteases, protease inhibitors, and other plasma membrane proteins, including the urokinase receptor (uPAR). LRP1 also functions in cell-signaling and regulates gene expression. The goal of this study was to determine whether LRP1 regulates remodeling of provisional extracellular matrix (ECM) by fibroblasts. To address this problem, we utilized an in vitro model in which type I collagen was reconstituted and overlaid with fibronectin. Either the collagen or fibronectin was fluorescently-labeled. ECM remodeling by fibroblasts deficient in LRP1, uPAR, or MT1-MMP was studied. MT1-MMP was required for efficient remodeling of the deep collagen layer but not involved in fibronectin remodeling. Instead, fibronectin was remodeled by a system that required urokinase-type plasminogen activator (uPA), uPAR, and exogenously-added plasminogen. LRP1 markedly inhibited fibronectin remodeling by regulating cell-surface uPAR and plasminogen activation. LRP1 also regulated remodeling of the deep collagen layer but not by controlling MT1-MMP. Instead, LRP1 deficiency or inhibition de-repressed a secondary pathway for collagen remodeling, which was active in MT1-MMP-deficient cells but not in uPAR-deficient cells. These results demonstrate that LRP1 regulates ECM remodeling principally by repressing pathways that require plasminogen activation by uPA in association with uPAR. PMID:19699300

  7. Micromechanical anisotropy and heterogeneity of the meniscus extracellular matrix.

    PubMed

    Li, Qing; Qu, Feini; Han, Biao; Wang, Chao; Li, Hao; Mauck, Robert L; Han, Lin

    2017-02-27

    To understand how the complex biomechanical functions of the meniscus are endowed by the nanostructure of its extracellular matrix (ECM), we studied the anisotropy and heterogeneity in the micromechanical properties of the meniscus ECM. We used atomic force microscopy (AFM) to quantify the time-dependent mechanical properties of juvenile bovine meniscus at deformation length scales corresponding to the diameters of collagen fibrils. At this scale, anisotropy in the elastic modulus of the circumferential fibers, the major ECM structural unit, can be attributed to differences in fibril deformation modes: uncrimping when normal to the fiber axis, and laterally constrained compression when parallel to the fiber axis. Heterogeneity among different structural units is mainly associated with their variations in microscale fiber orientation, while heterogeneity across anatomical zones is due to alterations in collagen fibril diameter and alignment at the nanoscale. Unlike the elastic modulus, the time-dependent properties are more homogeneous and isotropic throughout the ECM. These results enable a detailed understanding of the meniscus structure-mechanics at the nanoscale, and can serve as a benchmark for understanding meniscus biomechanical functions, documenting disease progression and designing tissue repair strategies.

  8. Conditional switches for extracellular matrix patterning in Drosophila melanogaster.

    PubMed

    Khokhar, Arvinder; Chen, Nan; Yuan, Ji-Ping; Li, Yishi; Landis, Gary N; Beaulieu, Gregory; Kaur, Harminder; Tower, John

    2008-03-01

    An F(1) mutagenesis strategy was developed to identify conditional mutations affecting extracellular matrix (ECM) patterning. Tubulogenesis requires coordinated movement of epithelial cells and deposition of a multilayered ECM. In the Drosophila ovary, an epithelium of follicle cells creates the eggshells, including the paired tubular dorsal appendages (DAs) that act as breathing tubes for the embryo. A P-element mutagenesis strategy allowed for conditional overexpression of hundreds of genes in follicle cells. Conditional phenotypes were scored at the level of individual mutant (F(1)) female flies. ECM pattern regulators were readily identified including MAPK signaling gene ets domain lacking (fused DAs), Wnt pathway genes frizzled 3 and osa (long DAs), Hh pathway gene debra (branched DAs), and transcription factor genes sima/HIF-1alpha, ush, lilli, Tfb1, broad, and foxo. In moving cells the [Ca(2+)]/calcineurin pathway can regulate adhesion to ECM while adherens junctions link cells together. Accordingly, thin eggshell and DA phenotypes were identified for the calcineurin regulator calreticulin and the adherens junction component arc. Finally a tubulogenesis defect phenotype was identified for the gene pterodactyl, homologous to the mammalian serine/threonine receptor-associated protein (STRAP) that integrates the TGF-beta and PI3K/AKT signaling pathways. Because phenotypes can be scored in each mutant fly before and after gene induction, this F(1) conditional mutagenesis strategy should allow for increased scale in screens for mutations affecting repeated (reiterated) events in adult animals, including gametogenesis, movement, behavior, and learning.

  9. Lung protection by inhalation of exogenous solubilized extracellular matrix

    PubMed Central

    Wu, Jinglei; Ravikumar, Priya; Nguyen, Kytai T.; Hsia, Connie C. W.

    2017-01-01

    Decellularized extracellular matrix (ECM) contains complex tissue-specific components that work in concert to promote tissue repair and constructive remodeling and has been used experimentally and clinically to accelerate epithelial wound repair, leading us to hypothesize that lung-derived ECM could mitigate acute lung injury. To explore the therapeutic potential of ECM for noninvasive delivery to the lung, we decellularized and solubilized porcine lung ECM, then characterized the composition, concentration, particle size and stability of the preparation. The ECM preparation at 3.2 mg/mL with average particle size <3 μm was tested in vitro on human A549 lung epithelial cells exposed to 95% O2 for 24 hours, and in vivo by tracheal instillation or nebulization into the lungs of rats exposed intermittently or continuously to 90% O2 for a cumulative 72 hours. Our results showed that the preparation was enriched in collagen, reduced in glycosaminoglycans, and contained various bioactive molecules. Particle size was concentration-dependent. Compared to the respective controls treated with cell culture medium in vitro or saline in vivo, ECM inhalation normalized cell survival and alveolar morphology, and reduced hyperoxia-induced apoptosis and oxidative damage. This proof-of-concept study established the methodology, feasibility and therapeutic potential of exogenous solubilized ECM for pulmonary cytoprotection, possibly as an adjunct or potentiator of conventional therapy. PMID:28151947

  10. Substrate stiffness regulates extracellular matrix deposition by alveolar epithelial cells

    PubMed Central

    Eisenberg, Jessica L; Safi, Asmahan; Wei, Xiaoding; Espinosa, Horacio D; Budinger, GR Scott; Takawira, Desire; Hopkinson, Susan B; Jones, Jonathan CR

    2012-01-01

    Aim The aim of the study was to address whether a stiff substrate, a model for pulmonary fibrosis, is responsible for inducing changes in the phenotype of alveolar epithelial cells (AEC) in the lung, including their deposition and organization of extracellular matrix (ECM) proteins. Methods Freshly isolated lung AEC from male Sprague Dawley rats were seeded onto polyacrylamide gel substrates of varying stiffness and analyzed for expression and organization of adhesion, cytoskeletal, differentiation, and ECM components by Western immunoblotting and confocal immunofluorescence microscopy. Results We observed that substrate stiffness influences cell morphology and the organization of focal adhesions and the actin cytoskeleton. Surprisingly, however, we found that substrate stiffness has no influence on the differentiation of type II into type I AEC, nor does increased substrate stiffness lead to an epithelial–mesenchymal transition. In contrast, our data indicate that substrate stiffness regulates the expression of the α3 laminin subunit by AEC and the organization of both fibronectin and laminin in their ECM. Conclusions An increase in substrate stiffness leads to enhanced laminin and fibronectin assembly into fibrils, which likely contributes to the disease phenotype in the fibrotic lung. PMID:23204878

  11. Extracellular matrix, mechanotransduction and structural hierarchies in heart tissue engineering.

    PubMed

    Parker, Kevin K; Ingber, Donald E

    2007-08-29

    The spatial and temporal scales of cardiac organogenesis and pathogenesis make engineering of artificial heart tissue a daunting challenge. The temporal scales range from nanosecond conformational changes responsible for ion channel opening to fibrillation which occurs over seconds and can lead to death. Spatial scales range from nanometre pore sizes in membrane channels and gap junctions to the metre length scale of the whole cardiovascular system in a living patient. Synchrony over these scales requires a hierarchy of control mechanisms that are governed by a single common principle: integration of structure and function. To ensure that the function of ion channels and contraction of muscle cells lead to changes in heart chamber volume, an elegant choreography of metabolic, electrical and mechanical events are executed by protein networks composed of extracellular matrix, transmembrane integrin receptors and cytoskeleton which are functionally connected across all size scales. These structural control networks are mechanoresponsive, and they process mechanical and chemical signals in a massively parallel fashion, while also serving as a bidirectional circuit for information flow. This review explores how these hierarchical structural networks regulate the form and function of living cells and tissues, as well as how microfabrication techniques can be used to probe this structural control mechanism that maintains metabolic supply, electrical activation and mechanical pumping of heart muscle. Through this process, we delineate various design principles that may be useful for engineering artificial heart tissue in the future.

  12. Three-dimensional reconstituted extracellular matrix scaffolds for tissue engineering.

    PubMed

    Narayanan, Karthikeyan; Leck, Kwong-Joo; Gao, Shujun; Wan, Andrew C A

    2009-09-01

    The extracellular matrix (ECM) is a rich meshwork of proteins and proteoglycans. Besides assuming a cell adhesive and structural support role, the ECM also helps to sequester and present growth factors to cells. ECM derived from tissues has been used as biological scaffolds for tissue engineering. In contrast, it has been difficult to employ ECM derived from cell lines as scaffolds due to its lack of form and structure. We have developed a mild, aqueous-based method for incorporating cell line derived ECM into biological scaffolds based on polyelectrolyte complexation, using the example of ECM from MC-3T3, a mouse preosteoblast cell line. A DNase step was incorporated in the ECM isolation procedure to further purify it of genetic material. Immunohistochemistry of fibers incorporated with MC-3T3 ECM reveal the presence of the ECM components, collagen type I, collagen type IV, fibronectin and heparan sulfate, on their surface. Reconstituted ECM scaffolds retained the cell-adhesion characteristics of the ECM, as demonstrated by 'reseeding' the ECM-secreting cell on the scaffolds. Human mesenchymal stem cells (hMSCs) were seeded onto the fibrous scaffolds incorporated with MC-3T3 ECM, and implanted subcutaneously into SCID mice. After 4 weeks of implantation, histological evidence showed that the hMSC seeded ECM scaffolds had induced bone formation at the ectopic site.

  13. Extracellular matrix content of ruptured anterior cruciate ligament tissue.

    PubMed

    Young, Kate; Samiric, Tom; Feller, Julian; Cook, Jill

    2011-08-01

    Anterior cruciate ligaments (ACLs) can rupture with simple movements, suggesting that structural changes in the ligament may reduce the loading capacity of the ligament. We aimed to investigate if proteoglycan and collagen levels were different between ruptured and non-ruptured ACLs. We also compared changes in ruptured tissue over time. During arthroscopic knee reconstruction surgery 24 ruptured ACLs were collected from participants (10 females; 14 males; mean age 24 years). Four non-ruptured ACLs were obtained from participants undergoing total knee replacement surgery (one female, three males; mean age 66 years). Western blot analysis was used to characterise core proteins of aggrecan, versican, decorin and biglycan and glycosaminoglycan assays were also conducted. Collagen levels were measured by hydroxyproline (OHPr) assays. Significantly lower levels of collagen, were found in ruptured ACL compared to non-ruptured ACL (p=0.004). Lower levels of both small and large proteoglycans were found in ruptured than non-ruptured ACLs. No correlation was found between time since rupture and proteoglycan or collagen levels. Ruptured ACLs had less collagen and proteoglycans than non-ruptured ACLs. These changes indicate either extracellular matrix protein levels were reduced prior to rupture or levels decreased immediately after rupture. It is possible that the composition and structure of ACLs that rupture are different to normal ACLs, potentially reducing the tissue's ability to withstand loading. An enhanced understanding of the aetiology of ACL injury could help identify individuals who may be predisposed to rupture.

  14. EVALUATION OF THE EXTRACELLULAR MATRIX OF INJURED SUPRASPINATUS IN RATS

    PubMed Central

    Almeida, Luiz Henrique Oliveira; Ikemoto, Roberto; Mader, Ana Maria; Pinhal, Maria Aparecida Silva; Munhoz, Bruna; Murachovsky, Joel

    2016-01-01

    ABSTRACT Objective: To evaluate the evolution of injuries of the supraspinatus muscle by immunohistochemistry (IHC) and anatomopathological analysis in animal model (Wistar rats). Methods: Twenty-five Wistar rats were submitted to complete injury of the supraspinatus tendon, then subsequently sacrificed in groups of five animals at the following periods: immediately after the injury, 24h after the injury, 48h after, 30 days after and three months after the injury. All groups underwent histological and IHC analysis. Results: Regarding vascular proliferation and inflammatory infiltrate, we found a statistically significant difference between groups 1(control group) and 2 (24h after injury). IHC analysis showed that expression of vascular endothelial growth factor (VEGF) showed a statistically significant difference between groups 1 and 2, and collagen type 1 (Col-1) evaluation presented a statistically significant difference between groups 1 and 4. Conclusion: We observed changes in the extracellular matrix components compatible with remodeling and healing. Remodeling is more intense 24h after injury. However, VEGF and Col-1 are substantially increased at 24h and 30 days after the injury, respectively. Level of Evidence I, Experimental Study. PMID:26997907

  15. Mining the extracellular matrix for tissue engineering applications.

    PubMed

    Pradhan, Swati; Farach-Carson, Mary C

    2010-11-01

    Tissue engineering is a rapidly evolving interdisciplinary field that aims to regenerate new tissue to replace damaged tissues or organs. The extracellular matrix (ECM) of animal tissues is a complex mixture of macromolecules that play an essential instructional role in the development of tissues and organs. Therefore, tissue engineering approaches rely on the need to present the correct cues to cells, to guide them to maintain tissue-specific functions. Recent research efforts have allowed us to mine various sequences and motifs, which play key roles in these guidance functions, from the ECM. Small conserved peptide sequences mined from ECM molecules can mimic some of the biological functions of their large parent molecules. In addition, these peptide sequences can be linked to various biomaterial scaffolds that can provide the cells with mechanical support to ensure appropriate cell growth and aid the formation of the correct tissue structure. The tissue engineering field will continue to benefit from the advent of these mined ECM sequences which have two major advantages over recombinant ECM molecules: material consistency and scalability.

  16. Adipose extracellular matrix remodelling in obesity and insulin resistance☆

    PubMed Central

    Lin, De; Chun, Tae-Hwa; Kang, Li

    2016-01-01

    The extracellular matrix (ECM) of adipose tissues undergoes constant remodelling to allow adipocytes and their precursor cells to change cell shape and function in adaptation to nutritional cues. Abnormal accumulation of ECM components and their modifiers in adipose tissues has been recently demonstrated to cause obesity-associated insulin resistance, a hallmark of type 2 diabetes. Integrins and other ECM receptors (e.g. CD44) that are expressed in adipose tissues have been shown to regulate insulin sensitivity. It is well understood that a hypoxic response is observed in adipose tissue expansion during obesity progression and that hypoxic response accelerates fibrosis and inflammation in white adipose tissues. The expansion of adipose tissues should require angiogenesis; however, the excess deposition of ECM limits the angiogenic response of white adipose tissues in obesity. While recent studies have focused on the metabolic consequences and the mechanisms of adipose tissue expansion and remodelling, little attention has been paid to the role played by the interaction between peri-adipocyte ECM and their cognate cell surface receptors. This review will address what is currently known about the roles played by adipose ECM, their modifiers, and ECM receptors in obesity and insulin resistance. Understanding how excess ECM deposition in the adipose tissue deteriorates insulin sensitivity would provide us hints to develop a new therapeutic strategy for the treatment of insulin resistance and type 2 diabetes. PMID:27179976

  17. Adipose extracellular matrix remodelling in obesity and insulin resistance.

    PubMed

    Lin, De; Chun, Tae-Hwa; Kang, Li

    2016-11-01

    The extracellular matrix (ECM) of adipose tissues undergoes constant remodelling to allow adipocytes and their precursor cells to change cell shape and function in adaptation to nutritional cues. Abnormal accumulation of ECM components and their modifiers in adipose tissues has been recently demonstrated to cause obesity-associated insulin resistance, a hallmark of type 2 diabetes. Integrins and other ECM receptors (e.g. CD44) that are expressed in adipose tissues have been shown to regulate insulin sensitivity. It is well understood that a hypoxic response is observed in adipose tissue expansion during obesity progression and that hypoxic response accelerates fibrosis and inflammation in white adipose tissues. The expansion of adipose tissues should require angiogenesis; however, the excess deposition of ECM limits the angiogenic response of white adipose tissues in obesity. While recent studies have focused on the metabolic consequences and the mechanisms of adipose tissue expansion and remodelling, little attention has been paid to the role played by the interaction between peri-adipocyte ECM and their cognate cell surface receptors. This review will address what is currently known about the roles played by adipose ECM, their modifiers, and ECM receptors in obesity and insulin resistance. Understanding how excess ECM deposition in the adipose tissue deteriorates insulin sensitivity would provide us hints to develop a new therapeutic strategy for the treatment of insulin resistance and type 2 diabetes. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Quercetin Attenuates Lactate Production and Extracellular Matrix Secretion in Keratoconus

    PubMed Central

    McKay, T. B.; Lyon, D.; Sarker-Nag, A.; Priyadarsini, S.; Asara, J. M.; Karamichos, D.

    2015-01-01

    Keratoconus(KC) is an ecstatic corneal disease leading to corneal-thinning and the formation of a cone-like cornea. Elevated lactate levels, increased oxidative stress, and myofibroblast formation have all been previously reported. In the current study, we assess the role of Quercetin on collagen secretion and myofibroblast formation in KC in vitro. Human corneal fibroblasts(HCFs) and human keratoconus cells(HKCs) were treated with a stable Vitamin C derivative and cultured for 4 weeks, stimulating formation of a self-assembled extracellular matrix. All samples were analyzed using Western blots and targeted tandem mass spectrometry. Our data showed that Quercetin significantly down regulates myofibroblast differentiation and fibrotic markers, such as α-smooth muscle actin (α-SMA) and Collagen III (Col III), in both HCFs and HKCs. Collagen III secretion was reduced 80% in both HCFs and HKCs following Quercetin treatment. Furthermore, Quercetin reduced lactate production by HKCs to normal HCF levels. Quercetin down regulated TGF-βR2 and TGF-β2 expression in HKCs suggesting a significant link to the TGF-β pathway. These results assert that Quercetin is a key regulator of fibrotic markers and ECM assembly by modulating cellular metabolism and TGF-β signaling. Our study suggests that Quercetin is a potential therapeutic for treatment of corneal dystrophies, such as KC. PMID:25758533

  19. Actin dynamics at sites of extracellular matrix degradation.

    PubMed

    Baldassarre, Massimiliano; Ayala, Inmaculada; Beznoussenko, Galina; Giacchetti, Giada; Machesky, Laura M; Luini, Alberto; Buccione, Roberto

    2006-12-01

    The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.

  20. Tissue-Specific Effects of Esophageal Extracellular Matrix

    PubMed Central

    Keane, Timothy J.; DeWard, Aaron; Londono, Ricardo; Saldin, Lindsey T.; Castleton, Arthur A.; Carey, Lisa; Nieponice, Alejandro; Lagasse, Eric

    2015-01-01

    Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate repair or remodeling of numerous tissues, including the esophagus. The theoretically ideal scaffold for tissue repair is the ECM derived from the particular tissue to be treated, that is, site-specific or homologous ECM. The preference or potential advantage for the use of site-specific ECM remains unknown in the esophageal location. The objective of the present study was to characterize the in vitro cellular response and in vivo host response to a homologous esophageal ECM (eECM) versus nonhomologous ECMs derived from small intestinal submucosa and urinary bladder. The in vitro response of esophageal stem cells was characterized by migration, proliferation, and three-dimensional (3D) organoid formation assays. The in vivo remodeling response was evaluated in a rat model of esophageal mucosal resection. Results of the study showed that the eECM retains favorable tissue-specific characteristics that enhance the migration of esophageal stem cells and supports the formation of 3D organoids to a greater extent than heterologous ECMs. Implantation of eECM facilitates the remodeling of esophageal mucosa following mucosal resection, but no distinct advantage versus heterologous ECM could be identified. PMID:26192009

  1. Tissue-Specific Effects of Esophageal Extracellular Matrix.

    PubMed

    Keane, Timothy J; DeWard, Aaron; Londono, Ricardo; Saldin, Lindsey T; Castleton, Arthur A; Carey, Lisa; Nieponice, Alejandro; Lagasse, Eric; Badylak, Stephen F

    2015-09-01

    Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate repair or remodeling of numerous tissues, including the esophagus. The theoretically ideal scaffold for tissue repair is the ECM derived from the particular tissue to be treated, that is, site-specific or homologous ECM. The preference or potential advantage for the use of site-specific ECM remains unknown in the esophageal location. The objective of the present study was to characterize the in vitro cellular response and in vivo host response to a homologous esophageal ECM (eECM) versus nonhomologous ECMs derived from small intestinal submucosa and urinary bladder. The in vitro response of esophageal stem cells was characterized by migration, proliferation, and three-dimensional (3D) organoid formation assays. The in vivo remodeling response was evaluated in a rat model of esophageal mucosal resection. Results of the study showed that the eECM retains favorable tissue-specific characteristics that enhance the migration of esophageal stem cells and supports the formation of 3D organoids to a greater extent than heterologous ECMs. Implantation of eECM facilitates the remodeling of esophageal mucosa following mucosal resection, but no distinct advantage versus heterologous ECM could be identified.

  2. Extracellular matrix protein CCN1 limits oncolytic efficacy in glioma.

    PubMed

    Haseley, Amy; Boone, Sean; Wojton, Jeffrey; Yu, Lianbo; Yoo, Ji Young; Yu, Jianhua; Kurozumi, Kazuhiko; Glorioso, Joseph C; Caligiuri, Michael A; Kaur, Balveen

    2012-03-15

    Oncolytic viral therapy has been explored widely as an option for glioma treatment but its effectiveness has remained limited. Cysteine rich 61 (CCN1) is an extracellular matrix (ECM) protein elevated in cancer cells that modulates their adhesion and migration by binding cell surface receptors. In this study, we examined a hypothesized role for CCN1 in limiting the efficacy of oncolytic viral therapy for glioma, based on evidence of CCN1 induction that occurs in this setting. Strikingly, we found that exogenous CCN1 in glioma ECM orchestrated a cellular antiviral response that reduced viral replication and limited cytolytic efficacy. Gene expression profiling and real-time PCR analysis revealed a significant induction of type-I interferon responsive genes in response to CCN1 exposure. This induction was accompanied by activation of the Jak/Stat signaling pathway, consistent with induction of an innate antiviral cellular response. Both effects were mediated by the binding of CCN1 to the cell surface integrin α6β1, activating its signaling and leading to rapid secretion of interferon-α, which was essential for the innate antiviral effect. Together, our findings reveal how an integrin signaling pathway mediates activation of a type-I antiviral interferon response that can limit the efficacy of oncolytic viral therapy. Furthermore, they suggest therapeutic interventions to inhibit CCN1-integrin α6 interactions to sensitize gliomas to viral oncolysis.

  3. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    PubMed

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  4. Extracellular matrix dynamics associated with tissue-engineered intravascular sclerotherapy.

    PubMed

    Vogel, Adam M; Smithers, C Jason; Kozakewich, Harry P; Zurakowski, David; Moses, Marsha A; Burrows, Patricia E; Fauza, Dario O; Fishman, Steven J

    2006-04-01

    The extracellular dynamics after intravascular sclerotherapy with an injectable, fibroblast-based engineered construct is unknown. Rabbits underwent ethanol sclerotherapy of a jugular vein segment. Control animals (n = 40) underwent no further treatment or an acellular collagen hydrogel was injected. Experimental animals (n = 20) received a tissue-engineered construct. After 1, 2, 4, and 20 to 24 weeks, segments were evaluated for collagen, glycosaminoglycan (GAG), matrix metalloproteinase (MMP) 2 and 9, and tissue inhibitors of MMP (TIMPs) 1 and 2 and scored on a scale of 0 to 3. Groups and time points were compared using nonparametric statistical analysis. Collagen content was higher in animals that received fibroblasts (P < .05). Glycosaminoglycan analysis showed a higher grade only at 1 week (P < .05). Collagen and GAG deposition were prominent at weeks 1 through 4, and decreased over time. Both MMP-2 and MMP-9 and TIMP-1 and TIMP-2 grade decreased with time (P < .01) in all groups, with no differences between groups. Enhancement of intravascular sclerotherapy by tissue engineering stems, at least in part, from increased local deposition of collagen and GAG. MMP and TIMPs may play a role in recanalization after experimental sclerotherapy. Tissue engineering may be a valuable adjunct for the treatment of vascular malformations.

  5. Cellular and extracellular matrix modulation of corneal stromal opacity

    PubMed Central

    Torricelli, Andre A. M.; Wilson, Steven E.

    2014-01-01

    Stromal transparency is a critical factor contributing to normal function of the visual system. Corneal injury, surgery, disease and infection elicit complex wound healing responses that serve to protect against insults and maintain the integrity of the cornea, and subsequently to restore corneal structure and transparency. However, in some cases these processes result in prolonged loss of corneal transparency and resulting diminished vision. Corneal opacity is mediated by the complex actions of many cytokines, growth factors, and chemokines produced by the epithelial cells, stromal cells, bone marrow-derived cells, lacrimal tissues, and nerves. Myofibroblasts, and the disorganized extracellular matrix produced by these cells, are critical determinants of the level and persistence of stromal opacity after corneal injury. Decreases in corneal crystallins in myofibroblasts and corneal fibroblasts contribute to cellular opacity in the stroma. Regeneration of a fully functional epithelial basement membrane (BM) appears to have a critical role in the maintenance of corneal stromal transparency after mild injuries and recovery of transparency when opacity is generated after severe injuries. The epithelial BM likely has a regulatory function whereby it modulates epithelium-derived growth factors such as transforming growth factor (TGF) β and platelet-derived growth factor (PDGF) that drive the development and persistence of myofibroblasts from precursor cells. The purpose of this article is to review the factors involved in the maintenance of corneal transparency and to highlight the mechanisms involved in the appearance, persistency and regression of corneal opacity after stromal injury. PMID:25281830

  6. Designing an extracellular matrix protein with enhanced mechanical stability

    PubMed Central

    Ng, Sean P.; Billings, Kate S.; Ohashi, Tomoo; Allen, Mark D.; Best, Robert B.; Randles, Lucy G.; Erickson, Harold P.; Clarke, Jane

    2007-01-01

    The extracellular matrix proteins tenascin and fibronectin experience significant mechanical forces in vivo. Both contain a number of tandem repeating homologous fibronectin type III (fnIII) domains, and atomic force microscopy experiments have demonstrated that the mechanical strength of these domains can vary significantly. Previous work has shown that mutations in the core of an fnIII domain from human tenascin (TNfn3) reduce the unfolding force of that domain significantly: The composition of the core is apparently crucial to the mechanical stability of these proteins. Based on these results, we have used rational redesign to increase the mechanical stability of the 10th fnIII domain of human fibronectin, FNfn10, which is directly involved in integrin binding. The hydrophobic core of FNfn10 was replaced with that of the homologous, mechanically stronger TNfn3 domain. Despite the extensive substitution, FNoTNc retains both the three-dimensional structure and the cell adhesion activity of FNfn10. Atomic force microscopy experiments reveal that the unfolding forces of the engineered protein FNoTNc increase by ≈20% to match those of TNfn3. Thus, we have specifically designed a protein with increased mechanical stability. Our results demonstrate that core engineering can be used to change the mechanical strength of proteins while retaining functional surface interactions. PMID:17535921

  7. Mixed Extracellular Matrix Ligands Synergistically Modulate Integrin Adhesion and Signaling

    PubMed Central

    Reyes, Catherine D.; Petrie, Timothy A.; García, Andrés J

    2008-01-01

    Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non-adhesive surfaces presenting well-defined, independent densities of two integrin-specific engineered ligands for the type I collagen (COL-I) receptor α2β1 and the fibronectin (FN) receptor α5β1 to evaluate the effects of integrin cross-talk on adhesive responses. Engineered surfaces displayed ligand density-dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL-I ligand surfaces. Moreover, surfaces presenting mixed COL-I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling. PMID:18613064

  8. Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae.

    PubMed

    Wu, Siva; Baum, Marc M; Kerwin, James; Guerrero, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-12-01

    Nontypeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen, can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24- and 96-h NTHi biofilms contained polysaccharides and proteinaceous components as detected by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24-h biofilms, two were found only in 96-h biofilms, and fifteen were present in the ECM of both 24- and 96-h NTHi biofilms. All proteins identified were either associated with bacterial membranes or cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation.

  9. Structural and biomechanical characterizations of porcine myocardial extracellular matrix

    PubMed Central

    Wang, Bo; Tedder, Mary E.; Perez, Clara E.; Wang, Guangjun; de Jongh Curry, Amy L.; To, Filip; Elder, Steven H.; Williams, Lakiesha N.; Simionescu, Dan T.; Liao, Jun

    2012-01-01

    Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson’s trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat’s pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine α-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regeneration and whole heart tissue engineering. PMID:22584822

  10. Brain extracellular matrix retains connectivity in neuronal networks.

    PubMed

    Bikbaev, Arthur; Frischknecht, Renato; Heine, Martin

    2015-09-29

    The formation and maintenance of connectivity are critically important for the processing and storage of information in neuronal networks. The brain extracellular matrix (ECM) appears during postnatal development and surrounds most neurons in the adult mammalian brain. Importantly, the removal of the ECM was shown to improve plasticity and post-traumatic recovery in the CNS, but little is known about the mechanisms. Here, we investigated the role of the ECM in the regulation of the network activity in dissociated hippocampal cultures grown on microelectrode arrays (MEAs). We found that enzymatic removal of the ECM in mature cultures led to transient enhancement of neuronal activity, but prevented disinhibition-induced hyperexcitability that was evident in age-matched control cultures with intact ECM. Furthermore, the ECM degradation followed by disinhibition strongly affected the network interaction so that it strongly resembled the juvenile pattern seen in naïve developing cultures. Taken together, our results demonstrate that the ECM plays an important role in retention of existing connectivity in mature neuronal networks that can be exerted through synaptic confinement of glutamate. On the other hand, removal of the ECM can play a permissive role in modification of connectivity and adaptive exploration of novel network architecture.

  11. The Biology of the Escherichia coli Extracellular Matrix

    PubMed Central

    Hufnagel, David A.; DePas, William H.; Chapman, Matthew R.

    2015-01-01

    Chapter Summary Escherichia coli (E. coli) is one of the world’s best-characterized organisms, as it has been extensively studied for over a century. However, most of this work has focused on E. coli grown under laboratory conditions that do not faithfully simulate its natural environments. Therefore, the historical perspectives on E. coli physiology and life cycle are somewhat skewed toward experimental systems that feature E. coli growing logarithmically in a test tube. Typically a commensal bacterium, E. coli resides in the lower intestines of a slew of animals. Outside of the lower intestine, E. coli can adapt and survive in a very different set of environmental conditions. Biofilm formation allows E. coli to survive, and even thrive, in environments that do not support the growth of planktonic populations. E. coli can form biofilms virtually everywhere; in the bladder during a urinary tract infection, on in-dwelling medical devices, and outside of the host on plants and in the soil. The E. coli extracellular matrix, primarily composed of the protein polymer named curli and the polysaccharide cellulose, promotes adherence to organic and inorganic surfaces, and resistance to desiccation, the host immune system and other antimicrobials. The pathways that govern E. coli biofilm formation, cellulose production, and curli biogenesis will be discussed in this book chapter, which concludes with insights into the future of E. coli biofilm research and potential therapies. PMID:26185090

  12. Fibroblast extracellular matrix and adhesion on microtextured polydimethylsiloxane scaffolds.

    PubMed

    Stanton, Morgan M; Parrillo, Allegra; Thomas, Gawain M; McGimpsey, W Grant; Wen, Qi; Bellin, Robert M; Lambert, Christopher R

    2015-05-01

    The immediate physical and chemical surroundings of cells provide important biochemical cues for their behavior. Designing and tailoring biomaterials for controlled cell signaling and extracellular matrix (ECM) can be difficult due to the complexity of the cell-surface relationship. To address this issue, our research has led to the development of a polydimethylsiloxane (PDMS) scaffold with defined microtopography and chemistry for surface driven ECM assembly. When human fibroblasts were cultured on this microtextured PDMS with 2-6 µm wide vertical features, significant changes in morphology, adhesion, actin cytoskeleton, and fibronectin generation were noted when compared with cells cultured on unmodified PDMS. Investigation of cellular response and behavior was performed with atomic force microscopy in conjunction with fluorescent labeling of focal adhesion cites and fibronectin in the ECM. Changes in the surface topography induced lower adhesion, an altered actin cytoskeleton, and compacted units of fibronectin similar to that observed in vivo. Overall, these findings provide critical information of cell-surface interactions with a microtextured, polymer substrate that can be used in the field of tissue engineering for controlling cellular ECM interactions.

  13. Brain extracellular matrix meets COST--matrix for European research networks.

    PubMed

    Gajović, Srećko; Pochet, Roland

    2014-01-01

    Today's researchers are faced with a change from curiosity-driven to mandate-driven research. These two approaches are well combined within scientific networks (Actions) supported by the European Cooperation in Science and Technology (COST) program. The functioning of COST Actions, although directed only to networking, has a substantial impact on European science and can be compared to the functioning of the extracellular matrix in the brain, which although scarce plays a key role in initiation, maintenance, and plasticity of intercellular interactions in the nervous system. COST networks enable interdisciplinary approach and support early-stage researchers, which is a vital asset for the advancement of science.

  14. Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques

    PubMed Central

    Langley, Sarah R.; Willeit, Karin; Didangelos, Athanasios; Matic, Ljubica Perisic; Skroblin, Philipp; Barallobre-Barreiro, Javier; Lengquist, Mariette; Rungger, Gregor; Kapustin, Alexander; Kedenko, Ludmilla; Molenaar, Chris; Lu, Ruifang; Barwari, Temo; Suna, Gonca; Iglseder, Bernhard; Paulweber, Bernhard; Willeit, Peter; Pasterkamp, Gerard; Davies, Alun H.; Monaco, Claudia; Hedin, Ulf; Shanahan, Catherine M.; Willeit, Johann; Kiechl, Stefan

    2017-01-01

    BACKGROUND. The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the “vulnerable plaque,” necessitating an improved approach for predicting onset of symptoms. METHODS. We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. RESULTS. Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. CONCLUSION. The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. FUNDING. UK: British Heart Foundation (BHF); King’s BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy’s and St

  15. Scatter factor binds to thrombospondin and other extracellular matrix components.

    PubMed Central

    Lamszus, K.; Joseph, A.; Jin, L.; Yao, Y.; Chowdhury, S.; Fuchs, A.; Polverini, P. J.; Goldberg, I. D.; Rosen, E. M.

    1996-01-01

    Scatter factor (SF) is an angiogenic growth factor that stimulates motility and invasion of carcinoma cells. SF is present in the extracellular matrix (ECM) of breast cancers, where it might act to promote tumor cell invasion and angiogenesis. To investigate how SF is incorporated into the ECM, we studied the binding of SF to various ECM components using a solid-phase binding assay based on the SF enzyme-linked immunosorbent assay. We found that SF binds to a variety of ECM molecules, with different binding capacities. The highest SF binding capacities were observed for thrombospondin-1 (TSP-1), fibronectin (Fn), and heparan sulfate proteoglycan, although SF did not bind to albumin. Mature two-chain SF and precursor single-chain SF bound approximately equally well to TSP-1 and Fn. Moreover, two SF alpha-chain peptides (NK1 and NK2) both bound to TSP-1 and Fn, suggesting that the whole SF molecule is not required for binding. Based on binding competition assays, TSP-1 exhibited higher affinity for SF than did nine other ECM molecules, including Fn and heparan sulfate proteoglycan. Although heparin in solution potently inhibited the binding of SF to TSP-1-coated surfaces, even very high concentrations of heparin could not elute SF already bound to TSP-1. SF binding was modulated by binding interactions among ECM molecules (TSP-1-Fn, TSP-1-collagen I, and Fn-collagen I), suggesting that the matrix capacity to bind SF depends upon its exact composition. SF bound in a dose-dependent fashion to ECMs secreted by three human breast carcinoma cell lines. Binding of SF to matrices from all three cell lines was significantly inhibited by preincubation of the matrices with antibodies against TSP-1, whereas antibodies against several other ECM components were less effective or ineffective in inhibiting SF binding. In addition, TSP-1 markedly inhibited chemotaxis of microvascular endothelial cells toward SF and SF-induced angiogenesis in the rat cornea neovascularization assay

  16. A new pre-loaded beam geometric stiffness matrix with full rigid body capabilities

    NASA Technical Reports Server (NTRS)

    Bosela, P. A.; Fertis, D. G.; Shaker, F. J.

    1992-01-01

    Space structures, such as the Space Station solar arrays, must be extremely light-weight, flexible structures. Accurate prediction of the natural frequencies and mode shapes is essential for determining the structural adequacy of components, and designing a controls system. The tension pre-load in the 'blanket' of photovoltaic solar collectors, and the free/free boundary conditions of a structure in space, causes serious reservations on the use of standard finite element techniques of solution. In particular, a phenomenon known as 'grounding', or false stiffening, of the stiffness matrix occurs during rigid body rotation. The authors have previously shown that the grounding phenomenon is caused by a lack of rigid body rotational capability, and is typical in beam geometric stiffness matrices formulated by others, including those which contain higher order effects. The cause of the problem was identified as the force imbalance inherent in the formulations. In this paper, the authors develop a beam geometric stiffness matrix for a directed force problem, and show that the resultant global stiffness matrix contains complete rigid body mode capabilities, and performs very well in the diagonalization methodology customarily used in dynamic analysis.

  17. Process-induced extracellular matrix alterations affect the mechanisms of soft tissue repair and regeneration

    PubMed Central

    Xu, Hui; Sandor, Maryellen; Lombardi, Jared

    2013-01-01

    Extracellular matrices derived from animal tissues for human tissue repairs are processed by various methods of physical, chemical, or enzymatic decellularization, viral inactivation, and terminal sterilization. The mechanisms of action in tissue repair vary among bioscaffolds and are suggested to be associated with process-induced extracellular matrix modifications. We compared three non-cross-linked, commercially available extracellular matrix scaffolds (Strattice, Veritas, and XenMatrix), and correlated extracellular matrix alterations to in vivo biological responses upon implantation in non-human primates. Structural evaluation showed significant differences in retaining native tissue extracellular matrix histology and ultrastructural features among bioscaffolds. Tissue processing may cause both the condensation of collagen fibers and fragmentation or separation of collagen bundles. Calorimetric analysis showed significant differences in the stability of bioscaffolds. The intrinsic denaturation temperature was measured to be 51°C, 38°C, and 44°C for Strattice, Veritas, and XenMatrix, respectively, demonstrating more extracellular matrix modifications in the Veritas and XenMatrix scaffolds. Consequently, the susceptibility to collagenase degradation was increased in Veritas and XenMatrix when compared to their respective source tissues. Using a non-human primate model, three bioscaffolds were found to elicit different biological responses, have distinct mechanisms of action, and yield various outcomes of tissue repair. Strattice permitted cell repopulation and was remodeled over 6 months. Veritas was unstable at body temperature, resulting in rapid absorption with moderate inflammation. XenMatrix caused severe inflammation and sustained immune reactions. This study demonstrates that extracellular matrix alterations significantly affect biological responses in soft tissue repair and regeneration. The data offer useful insights into the rational design of

  18. MatrixDB, the extracellular matrix interaction database: updated content, a new navigator and expanded functionalities.

    PubMed

    Launay, G; Salza, R; Multedo, D; Thierry-Mieg, N; Ricard-Blum, S

    2015-01-01

    MatrixDB (http://matrixdb.ibcp.fr) is a freely available database focused on interactions established by extracellular proteins and polysaccharides. It is an active member of the International Molecular Exchange (IMEx) consortium and has adopted the PSI-MI standards for annotating and exchanging interaction data, either at the MIMIx or IMEx level. MatrixDB content has been updated by curation and by importing extracellular interaction data from other IMEx databases. Other major changes include the creation of a new website and the development of a novel graphical navigator, iNavigator, to build and expand interaction networks. Filters may be applied to build sub-networks based on a list of biomolecules, a specified interaction detection method and/or an expression level by tissue, developmental stage, and health state (UniGene data). Any molecule of the network may be selected and its partners added to the network at any time. Networks may be exported under Cytoscape and tabular formats and as images, and may be saved for subsequent re-use. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation.

    PubMed

    Oliveira, Marina C; Arntz, Onno J; Blaney Davidson, Esmeralda N; van Lent, Peter L E M; Koenders, Marije I; van der Kraan, Peter M; van den Berg, Wim B; Ferreira, Adaliene V M; van de Loo, Fons A J

    2016-04-01

    The claimed beneficial effect of milk on bone is still a matter for debate. Recently extracellular vesicles (EVs) that contain proteins and RNA were discovered in milk, but their effect on bone formation has not yet been determined. We demonstrated previously that bovine milk-derived EVs (BMEVs) have immunoregulatory properties. Our aim was to evaluate the effect of BMEVs on osteogenesis by mice and human mesenchymal stem cells (hMSCs). Oral delivery of two concentrations of BMEVs to female DBA/1J mice during 7weeks did not alter the tibia trabecular bone area; however, the osteocytes number increased. In addition, the highest dose of BMEVs markedly increased the woven bone tissue, which is more brittle. The exposure of hMSCs to BMEVs during 21days resulted in less mineralization but higher cell proliferation. Interestingly BMEVs reduced the collagen production, but enhanced the expression of genes characteristic for immature osteoblasts. A kinetic study showed that BMEVs up-regulated many osteogenic genes within the first 4days. However, the production of type I collagen and expression of its genes (COL1A1 and COL1A2) were markedly reduced at days 21 and 28. At day 28, BMEVs again lead to higher proliferation, but mineralization was significantly increased. This was associated with increased expression of sclerostin, a marker for osteocytes, and reduced osteonectin, which is associated to bone matrix formation. Our study adds BMEVs to the list of milk components that can affect bone formation and may shed new light on the contradictory claims of milk on bone formation. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis.

    PubMed

    d'Amaro, Rocca; Scheidegger, Rolf; Blumer, Susan; Pazera, Pawel; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf

  1. An innovative protocol for schwann cells extracellular matrix proteins extraction.

    PubMed

    Parisi, L; Zomer Volpato, F; Cagol, N; Siciliano, M; Migliaresi, C; Motta, A; Sala, R

    2016-12-01

    The evidence that extracellular matrix (ECM) components could represent new targets for drugs designed to approach degenerative disease, requires their analysis. Before the analysis, proteins should be extracted from ECM and solubilized. Currently, few protocols for ECM proteins extraction and solubilization are available in literature, and most of them are based mainly on the use of proteolytic enzymes, such as trypsin, which often lead to proteins damage. Moreover, no methods have been so far proposed to solubilize Schwann Cell ECM, which may represent an important target for the therapy of neurodegenerative disorders. In our study, we propose to solubilize SC ECM through the use of surfactants and urea. We compared our method of solubilization, with one of that proposed in literature for a general ECM, mainly based on the use of enzymes. We want to highlight the benefit of solubilizing SC ECM, avoiding the use of proteolytic enzymes. To compare the amount of proteins extracted with both methods, MicroBCA assay was used, while the quality of the proteins extracted was observed through the SDS-PAGE. The results obtained confirm a better solubilization of SC ECM proteins with the proposed protocol, both quantitatively and qualitatively, showing a higher concentration of proteins extracted and a better enrichment of protein fractions, if compared to the enzyme-based protocol. Our results show that SC ECM could be efficiently solubilized through the use of surfactant and urea, avoiding the use of enzyme-base methods. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3175-3180, 2016.

  2. Extracellular Matrix-Mediated Differentiation of Periodontal Progenitor Cells

    PubMed Central

    Dangaria, Smit J.; Ito, Yoshihiro; Walker, Cameron; Druzinsky, Robert; Luan, Xianghong; Diekwisch, Thomas G.H.

    2009-01-01

    The periodontal ligament (PDL) is a specialized connective tissue that connects the surface of the tooth root with the bony tooth socket. The healthy PDL harbors stem cell niches and extracellular matrix (ECM) microenvironments that facilitate periodontal regeneration. During periodontal disease, the PDL is often compromised or destroyed, reducing the life-span of the tooth. In order to explore new approaches toward the regeneration of diseased periodontal tissues, we have tested the effect of periodontal ECM signals, fibroblast growth factor 2 (FGF2), connective tissue growth factor (CTGF), and the cell adhesion peptide Arg-Gly- Asp (RGD) on the differentiation of two types of periodontal progenitor cells, PDL progenitor cells (PDLPs) and dental follicle progenitor cells (DFCs). Our studies documented that CTGF and FGF2 significantly enhanced the expression of collagens I & III, biglycan and periostin in tissue engineered regenerates after 4 weeks compared to untreated controls. Specifically, CTGF promoted mature PDL-like tissue regeneration as demonstrated by dense periostin localization in collagen fiber bundles. CTGF and FGF2 displayed synergistic effects on collagen III and biglycan gene expression, while effects on mineralization were antagonistic to each other: CTGF promoted while FGF2 inhibited mineralization in PDL cell cultures. Incorporation of RGD peptides in hydrogel matrices significantly enhanced attachment, spreading, survival and mineralization of the encapsulated DFCs, suggesting that RGD additives might promote the use of hydrogels for periodontal mineralized tissue engineering. Together, our studies have documented the effect of three key components of the periodontal ECM on the differentiation of periodontal progenitor populations. PMID:19433344

  3. Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration

    PubMed Central

    Chen, William C. W.; Wang, Zhouguang; Missinato, Maria Azzurra; Park, Dae Woo; Long, Daniel Ward; Liu, Heng-Jui; Zeng, Xuemei; Yates, Nathan A.; Kim, Kang; Wang, Yadong

    2016-01-01

    Heart attack is a global health problem that leads to significant morbidity, mortality, and health care burden. Adult human hearts have very limited regenerative capability after injury. However, evolutionarily primitive species generally have higher regenerative capacity than mammals. The extracellular matrix (ECM) may contribute to this difference. Mammalian cardiac ECM may not be optimally inductive for cardiac regeneration because of the fibrotic, instead of regenerative, responses in injured adult mammalian hearts. Given the high regenerative capacity of adult zebrafish hearts, we hypothesize that decellularized zebrafish cardiac ECM (zECM) made from normal or healing hearts can induce mammalian heart regeneration. Using zebrafish and mice as representative species of lower vertebrates and mammals, we show that a single administration of zECM, particularly the healing variety, enables cardiac functional recovery and regeneration of adult mouse heart tissues after acute myocardial infarction. zECM-treated groups exhibit proliferation of the remaining cardiomyocytes and multiple cardiac precursor cell populations and reactivation of ErbB2 expression in cardiomyocytes. Furthermore, zECM exhibits pro-proliferative and chemotactic effects on human cardiac precursor cell populations in vitro. These contribute to the structural preservation and correlate with significantly higher cardiac contractile function, notably less left ventricular dilatation, and substantially more elastic myocardium in zECM-treated hearts than control animals treated with saline or decellularized adult mouse cardiac ECM. Inhibition of ErbB2 activity abrogates beneficial effects of zECM administration, indicating the possible involvement of ErbB2 signaling in zECM-mediated regeneration. This study departs from conventional focuses on mammalian ECM and introduces a new approach for cardiac tissue regeneration. PMID:28138518

  4. Extracellular matrix and sex-inducing pheromone in Volvox.

    PubMed

    Hallmann, Armin

    2003-01-01

    During evolution of multicellularity it was imperative to create a complex, multifunctional extracellular matrix (ECM) out of the simple cell wall of a unicellular ancestor. The green alga Volvox represents one of the simplest multicellular organisms, but even so, it already has a highly developed ECM. This ECM is mainly composed of an assortment of glycoproteins, many of which are hydroxyproline rich and extensively sulfated. Several ECM proteins are cross-linked and might have only structural functions. However, the ECM does not represent a static but rather a dynamic and multifunctional interface between a cell and its neighboring cells or its environment. It not only provides protection and structural support for the shape of each cell and the organism as a whole, but also plays a broad range of biological roles in growth, development, reproduction, and responses to environmental stress or wounding. The variety of functions of the ECM requires many glycoproteins to do the work. To attain a high flexibility and adaptability, almost all ECM glycoproteins from Volvox consist of modules, defined as functional subunits that form modular mosaic proteins with an outstanding combinatorial potential. The ECM's functions are not only extensive but also change under developmental control or by environmental incidents. The changing scope of duties necessitates a permanent ECM turnover and remodeling. In Volvox carteri one particularly challenging trigger of such ECM modifications is a sex-inducing pheromone, which is one of the most potent biological effector molecules known: the glycoprotein pheromone is fully effective for inducing sexual development in males and females at concentrations as low as 10(-16) M. The earliest detectable response to the pheromone is the synthesis of ECM glycoproteins.

  5. Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound

    PubMed Central

    Tracy, Lauren E.; Minasian, Raquel A.; Caterson, E.J.

    2016-01-01

    Significance: Fibroblasts play a critical role in normal wound healing. Various extracellular matrix (ECM) components, including collagens, fibrin, fibronectin, proteoglycans, glycosaminoglycans, and matricellular proteins, can be considered potent protagonists of fibroblast survival, migration, and metabolism. Recent Advances: Advances in tissue culture, tissue engineering, and ex vivo models have made the examination and precise measurements of ECM components in wound healing possible. Likewise, the development of specific transgenic animal models has created the opportunity to characterize the role of various ECM molecules in healing wounds. In addition, the recent characterization of new ECM molecules, including matricellular proteins, dermatopontin, and FACIT collagens (Fibril-Associated Collagens with Interrupted Triple helices), further demonstrates our cursory knowledge of the ECM in coordinated wound healing. Critical Issues: The manipulation and augmentation of ECM components in the healing wound is emerging in patient care, as demonstrated by the use of acellular dermal matrices, tissue scaffolds, and wound dressings or topical products bearing ECM proteins such as collagen, hyaluronan (HA), or elastin. Once thought of as neutral structural proteins, these molecules are now known to directly influence many aspects of cellular wound healing. Future Directions: The role that ECM molecules, such as CCN2, osteopontin, and secreted protein, acidic and rich in cysteine, play in signaling homing of fibroblast progenitor cells to sites of injury invites future research as we continue investigating the heterotopic origin of certain populations of fibroblasts in a healing wound. Likewise, research into differently sized fragments of the same polymeric ECM molecule is warranted as we learn that fragments of molecules such as HA and tenascin-C can have opposing effects on dermal fibroblasts. PMID:26989578

  6. Expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinases during mouse embryonic development.

    PubMed

    Chen, Li; Nakai, Masaaki; Belton, Robert J; Nowak, Romana A

    2007-02-01

    Mouse embryo implantation is a highly invasive and controlled process that involves remodeling and degradation of the extracellular matrix of the uterus. Matrix metalloproteinases (MMPs) are the main proteinases facilitating this process. Extracellular matrix metalloproteinase inducer (EMMPRIN) can stimulate the production of MMPs and is required for successful implantation in the mouse. The aims of the present study were to examine the expression profiles of mRNA and proteins for EMMPRIN and MMPs in the developing mouse embryo in vitro, and to study whether EMMPRIN protein induces the production of MMPs by mouse blastocysts. EMMPRIN mRNA, detected by RT-PCR, was present at all stages of embryo development from the one-cell to the blastocyst outgrowth. EMMPRIN protein, observed by confocal microscopy, was present on the cell surface at the same stages of development as was the mRNA. Of seven MMPs studied, murine collagenase-like A (Mcol-A), murine collagenase-like B (Mcol-B) and gelatinase A (MMP-2) mRNAs were detected only in blastocyst outgrowths by RT-PCR. Gelatinase B (MMP-9) mRNA was detected both in expanded blastocysts and blastocyst outgrowths. MMP-2 and -9 proteins were detected in the cytoplasm of outgrowing trophoblast cells. Collagenase-2 (MMP-8), collagenase-3 (MMP-13), or stromelysin-1 (MMP-3) mRNAs were not present at any stage of pre- or peri-implantation mouse embryo development. Quantitative RT-PCR analyses showed that recombinant EMMPRIN protein did not stimulate MMP-2 or -9 expression by mouse blastocyst outgrowths. These data suggest that EMMPRIN may regulate physiological functions other than MMP production by mouse embryos during implantation.

  7. Extracellular matrix elasticity modulates TGF-β-induced p38 activation and myofibroblast transdifferentiation in human tenon fibroblasts.

    PubMed

    Meyer-ter-Vehn, Tobias; Han, Hong; Grehn, Franz; Schlunck, Günther

    2011-11-25

    Extracellular matrix and the cytokine TGF-β influence scar formation in an interdependent fashion. In this study, the impact of extracellular matrix elasticity on TGF-β-induced signal transduction and myofibroblast transdifferentiation was examined. Primary human tenon fibroblasts were seeded on collagen-coated glass coverslips (rigid environment) or collagen or polyacrylamide gels (elastic environment) of different compliance and stimulated with TGF-β. Myofibroblast transdifferentiation was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis for the marker gene α-smooth muscle actin (SMA), and SMA incorporation into stress fibers was determined by confocal immunofluorescence microscopy. CTGF transcription was assessed by RT-qPCR. Signaling pathways were examined by Western blot using phosphospecific antibodies and by immunofluorescence microscopy. TGF-β-dependent myofibroblast transdifferentiation was enhanced in a stiff environment. Increasing matrix elasticity attenuated TGF-β-induced myofibroblast transdifferentiation and the associated CTGF expression. TGF-β-induced p38 activation was reduced on elastic substrates. The results suggest that matrix elasticity influences TGF-β-dependent activation of p38 signaling and subsequent myofibroblast transdifferentiation. Biomechanical cues represent an important determinant of scarring processes. Therefore, cellular signals elicited by mechanotransduction deserve consideration in the design of novel antifibrotic strategies.

  8. Force-induced elastic matrix-mediated interactions in the presence of a rigid wall.

    PubMed

    Menzel, Andreas M

    2017-05-14

    We consider a soft elastic matrix that contains particulate inclusions and is bounded by a rigid wall, e.g., the substrate. Such a situation arises in elastic composite materials. They may serve as soft actuators when forces are imposed on or induced between the embedded particles. We investigate how the presence of the rigid wall affects the interactions between the inclusions. First, for no-slip boundary conditions, we transfer Blake's derivation of a corresponding Green's function from low-Reynolds-number hydrodynamics to the linearly elastic case. Then, we list the general expressions to describe the situation for point-like particles in the presence of no-slip and free-slip surface conditions. To compare the effect of the different surface conditions to each other and to the bulk behavior, we address the example situation of pairwise interactions between two embedded particles. The axis through both particle centers is either aligned parallel or perpendicular to the surface. Our results suggest that walls with free-slip surface conditions are preferred when they serve as substrates for soft actuators made from elastic composite materials. As we further demonstrate, the presence of a rigid wall can qualitatively change the interactions between the inclusions. In effect, it can switch attractive interactions into repulsive ones (and vice versa). It should be straightforward to observe the effects in future experiments and to combine our results, e.g., with the modeling of biological cells and tissue on rigid surfaces.

  9. Matrix Rigidity Activates Wnt Signaling through Down-regulation of Dickkopf-1 Protein*

    PubMed Central

    Barbolina, Maria V.; Liu, Yiuying; Gurler, Hilal; Kim, Mijung; Kajdacsy-Balla, Andre A.; Rooper, Lisa; Shepard, Jaclyn; Weiss, Michael; Shea, Lonnie D.; Penzes, Peter; Ravosa, Matthew J.; Stack, M. Sharon

    2013-01-01

    Cells respond to changes in the physical properties of the extracellular matrix with altered behavior and gene expression, highlighting the important role of the microenvironment in the regulation of cell function. In the current study, culture of epithelial ovarian cancer cells on three-dimensional collagen I gels led to a dramatic down-regulation of the Wnt signaling inhibitor dickkopf-1 with a concomitant increase in nuclear β-catenin and enhanced β-catenin/Tcf/Lef transcriptional activity. Increased three-dimensional collagen gel invasion was accompanied by transcriptional up-regulation of the membrane-tethered collagenase membrane type 1 matrix metalloproteinase, and an inverse relationship between dickkopf-1 and membrane type 1 matrix metalloproteinase was observed in human epithelial ovarian cancer specimens. Similar results were obtained in other tissue-invasive cells such as vascular endothelial cells, suggesting a novel mechanism for functional coupling of matrix adhesion with Wnt signaling. PMID:23152495

  10. Matrix rigidity activates Wnt signaling through down-regulation of Dickkopf-1 protein.

    PubMed

    Barbolina, Maria V; Liu, Yiuying; Gurler, Hilal; Kim, Mijung; Kajdacsy-Balla, Andre A; Rooper, Lisa; Shepard, Jaclyn; Weiss, Michael; Shea, Lonnie D; Penzes, Peter; Ravosa, Matthew J; Stack, M Sharon

    2013-01-04

    Cells respond to changes in the physical properties of the extracellular matrix with altered behavior and gene expression, highlighting the important role of the microenvironment in the regulation of cell function. In the current study, culture of epithelial ovarian cancer cells on three-dimensional collagen I gels led to a dramatic down-regulation of the Wnt signaling inhibitor dickkopf-1 with a concomitant increase in nuclear β-catenin and enhanced β-catenin/Tcf/Lef transcriptional activity. Increased three-dimensional collagen gel invasion was accompanied by transcriptional up-regulation of the membrane-tethered collagenase membrane type 1 matrix metalloproteinase, and an inverse relationship between dickkopf-1 and membrane type 1 matrix metalloproteinase was observed in human epithelial ovarian cancer specimens. Similar results were obtained in other tissue-invasive cells such as vascular endothelial cells, suggesting a novel mechanism for functional coupling of matrix adhesion with Wnt signaling.

  11. Targeting the extracellular matrix: matricellular proteins regulate cell-extracellular matrix communication within distinct niches of the intervertebral disc.

    PubMed

    Bedore, Jake; Leask, Andrew; Séguin, Cheryle A

    2014-07-01

    The so-called "matricellular" proteins have recently emerged as important regulators of cell-extracellular matrix (ECM) interactions. These proteins modulate a variety of cell functions through a range of interactions with cell-surface receptors, hormones, proteases and structural components of the ECM. As such, matricellular proteins are crucial regulators of cell phenotype, and consequently tissue function. The distinct cell types and microenvironments that together form the IVD provide an excellent paradigm to study how matricellular proteins mediate communication within and between adjacent tissue types. In recent years, the role of several matricellular proteins in the intervertebral disc has been explored in vivo using mutant mouse models in which the expression of target matricellular proteins was deleted from either one or all compartments of the intervertebral disc. The current review outlines what is presently known about the roles of the matricellular proteins belonging to the CCN family, SPARC (Secreted Protein, Acidic, and Rich in Cysteine), and thrombospondin (TSP) 2 in regulating intervertebral disc cell-ECM interactions, ECM synthesis and disc tissue homeostasis using genetically modified mouse models. Furthermore, we provide a brief overview of recent preliminary studies of other matricellular proteins including, periostin (POSTN) and tenascin (TN). Each specific tissue type of the IVD contains a different matricellular protein signature, which varies based on the specific stage of development, maturity or disease. A growing body of direct genetic evidence links IVD development, maintenance and repair to the coordinate interaction of matricellular proteins within their respective niches and suggests that several of these signaling modulators hold promise in the development of diagnostics and/or therapeutics targeting intervertebral disc aging and/or degeneration.

  12. Supercritical carbon dioxide extracted extracellular matrix material from adipose tissue.

    PubMed

    Wang, Jun Kit; Luo, Baiwen; Guneta, Vipra; Li, Liang; Foo, Selin Ee Min; Dai, Yun; Tan, Timothy Thatt Yang; Tan, Nguan Soon; Choong, Cleo; Wong, Marcus Thien Chong

    2017-06-01

    Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO2) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO2-treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO2-treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO2-treated ECM coating can be potentially used for various biomedical applications. The SC-CO2-treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO2-treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO2-treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall, this study shows the efficacy of SC-CO2

  13. Adapted Boolean network models for extracellular matrix formation

    PubMed Central

    Wollbold, Johannes; Huber, René; Pohlers, Dirk; Koczan, Dirk; Guthke, Reinhard; Kinne, Raimund W; Gausmann, Ulrike

    2009-01-01

    Background Due to the rapid data accumulation on pathogenesis and progression of chronic inflammation, there is an increasing demand for approaches to analyse the underlying regulatory networks. For example, rheumatoid arthritis (RA) is a chronic inflammatory disease, characterised by joint destruction and perpetuated by activated synovial fibroblasts (SFB). These abnormally express and/or secrete pro-inflammatory cytokines, collagens causing joint fibrosis, or tissue-degrading enzymes resulting in destruction of the extra-cellular matrix (ECM). We applied three methods to analyse ECM regulation: data discretisation to filter out noise and to reduce complexity, Boolean network construction to implement logic relationships, and formal concept analysis (FCA) for the formation of minimal, but complete rule sets from the data. Results First, we extracted literature information to develop an interaction network containing 18 genes representing ECM formation and destruction. Subsequently, we constructed an asynchronous Boolean network with biologically plausible time intervals for mRNA and protein production, secretion, and inactivation. Experimental gene expression data was obtained from SFB stimulated by TGFβ1 or by TNFα and discretised thereafter. The Boolean functions of the initial network were improved iteratively by the comparison of the simulation runs to the experimental data and by exploitation of expert knowledge. This resulted in adapted networks for both cytokine stimulation conditions. The simulations were further analysed by the attribute exploration algorithm of FCA, integrating the observed time series in a fine-tuned and automated manner. The resulting temporal rules yielded new contributions to controversially discussed aspects of fibroblast biology (e.g., considerable expression of TNF and MMP9 by fibroblasts stimulation) and corroborated previously known facts (e.g., co-expression of collagens and MMPs after TNFα stimulation), but also revealed

  14. Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

    PubMed Central

    d'Amaro, Rocca; Scheidegger, Rolf; Blumer, Susan; Pazera, Pawel; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf

  15. Mechanics and crack formation in the extracellular matrix with articular cartilage as a model system

    NASA Astrophysics Data System (ADS)

    Kearns, Sarah; Silverberg, Jesse; Bonassar, Lawrence; Cohen, Itai; Das, Moumita

    We investigate the mechanical structure-function relations in the extracellular matrix (ECM) with focus on crack formation and failure. As a model system, our study focuses on the ECM in articular cartilage (AC), the tissue that covers the ends of bones, and distributes load in joints including in the knees, shoulders, and hips. The strength, toughness, and crack resistance of native articular cartilage is unparalleled in materials made by humankind. This mechanical response is mainly due to its ECM. The ECM in AC has two major mechanobiological components: a network of the biopolymer collagen and a flexible aggrecan gel. We model this system as a biopolymer network embedded in a swelling gel, and investigate the conditions for the formation and propagation of cracks using a combination of rigidity percolation theory and energy minimization approaches. Our results may provide useful insights into the design principles of the ECM as well as of biomimetic hydrogels that are mechanically robust and can, at the same time, easily adapt to cues in their surroundings. This work was partially supported by a Cottrell College Science Award.

  16. Engineering strategies to recapitulate epithelial morphogenesis within synthetic 3 dimensional extracellular matrix with tunable mechanical properties

    PubMed Central

    Miroshnikova, Y.A.; Jorgens, D.M.; Spirio, L.; Auer, M.; Sieminski-Sarang, A.L.; Weaver, V.M.

    2011-01-01

    The mechanical properties (e.g. stiffness) of the extracellular matrix (ECM) influence cell fate and tissue morphogenesis and contribute to disease progression. Nevertheless, our understanding of the mechanisms by which ECM rigidity modulates cell behavior and fate remains rudimentary. To address this issue, a number of two and three dimensional (3D) hydrogel systems have been used to explore the effects of mechanical properties of the ECM on cell behavior. Unfortunately, many of these systems have limited application because fiber architecture, adhesiveness and/or pore size often change in parallel when gel elasticity is varied. Here we describe the use of ECM-adsorbed, synthetic, self-assembling peptide gels (SAPs) that are able to recapitulate normal epithelial acini morphogenesis and gene expression in a 3D context. By exploiting the range of visco-elasticity attainable with these SAP gels, and their ability to recreate native-like ECM fibril topology with minimal variability in ligand density and pore size, we were able to reconstitute normal versus tumor-like phenotype and gene expression patterns in nonmalignant mammary epithelial cells (MECs). Accordingly, this SAP hydrogel system presents the first tunable system capable of independently assessing the interplay between ECM stiffness and multi-cellular epithelial phenotype in a 3D context. PMID:21441648

  17. Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins

    PubMed Central

    Barallobre-Barreiro, Javier; Oklu, Rahmi; Lynch, Marc; Fava, Marika; Baig, Ferheen; Yin, Xiaoke; Barwari, Temo; Potier, David N.; Albadawi, Hassan; Jahangiri, Marjan; Porter, Karen E.; Watkins, Michael T.; Misra, Sanjay; Stoughton, Julianne; Mayr, Manuel

    2016-01-01

    Aims Extracellular matrix remodelling has been implicated in a number of vascular conditions, including venous hypertension and varicose veins. However, to date, no systematic analysis of matrix remodelling in human veins has been performed. Methods and results To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix. Varicose saphenous veins removed during phlebectomy and normal saphenous veins obtained during coronary artery bypass surgery were collected for proteomics analysis. Extracellular matrix proteins were enriched from venous tissues. The proteomics analysis revealed the presence of >150 extracellular matrix proteins, of which 48 had not been previously detected in venous tissue. Extracellular matrix remodelling in varicose veins was characterized by a loss of aggrecan and several small leucine-rich proteoglycans and a compensatory increase in collagen I and laminins. Gene expression analysis of the same tissues suggested that the remodelling process associated with venous hypertension predominantly occurs at the protein rather than the transcript level. The loss of aggrecan in varicose veins was paralleled by a reduced expression of aggrecanases. Chymase and tryptase β1 were among the up-regulated proteases. The effect of these serine proteases on the venous extracellular matrix was further explored by incubating normal saphenous veins with recombinant enzymes. Proteomics analysis revealed extensive extracellular matrix degradation after digestion with tryptase β1. In comparison, chymase was less potent and degraded predominantly basement membrane-associated proteins. Conclusion The present proteomics study provides unprecedented insights into the expression and degradation of structural and regulatory components of the vascular extracellular matrix in varicosis. PMID:27068509

  18. Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins.

    PubMed

    Barallobre-Barreiro, Javier; Oklu, Rahmi; Lynch, Marc; Fava, Marika; Baig, Ferheen; Yin, Xiaoke; Barwari, Temo; Potier, David N; Albadawi, Hassan; Jahangiri, Marjan; Porter, Karen E; Watkins, Michael T; Misra, Sanjay; Stoughton, Julianne; Mayr, Manuel

    2016-06-01

    Extracellular matrix remodelling has been implicated in a number of vascular conditions, including venous hypertension and varicose veins. However, to date, no systematic analysis of matrix remodelling in human veins has been performed. To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix. Varicose saphenous veins removed during phlebectomy and normal saphenous veins obtained during coronary artery bypass surgery were collected for proteomics analysis. Extracellular matrix proteins were enriched from venous tissues. The proteomics analysis revealed the presence of >150 extracellular matrix proteins, of which 48 had not been previously detected in venous tissue. Extracellular matrix remodelling in varicose veins was characterized by a loss of aggrecan and several small leucine-rich proteoglycans and a compensatory increase in collagen I and laminins. Gene expression analysis of the same tissues suggested that the remodelling process associated with venous hypertension predominantly occurs at the protein rather than the transcript level. The loss of aggrecan in varicose veins was paralleled by a reduced expression of aggrecanases. Chymase and tryptase β1 were among the up-regulated proteases. The effect of these serine proteases on the venous extracellular matrix was further explored by incubating normal saphenous veins with recombinant enzymes. Proteomics analysis revealed extensive extracellular matrix degradation after digestion with tryptase β1. In comparison, chymase was less potent and degraded predominantly basement membrane-associated proteins. The present proteomics study provides unprecedented insights into the expression and degradation of structural and regulatory components of the vascular extracellular matrix in varicosis. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.

  19. Extracellular matrix directions estimation of the heart on micro-focus x-ray CT volumes

    NASA Astrophysics Data System (ADS)

    Oda, Hirohisa; Oda, Masahiro; Kitasaka, Takayuki; Akita, Toshiaki; Mori, Kensaku

    2017-03-01

    In this paper we propose an estimation method of extracellular matrix directions of the heart. Myofiber are surrounded by the myocardial cell sheets whose directions have strong correspondence between heart failure. Estimation of the myocardial cell sheet directions is difficult since they are very thin. Therefore, we estimate the extracellular matrices which are touching to the sheets as if piled up. First, we perform a segmentation of the extracellular matrices by using the Hessian analysis. Each extracellular matrix region has sheet-like shape. We estimate the direction of each extracellular matrix region by the principal component analysis (PCA). In our experiments, mean inclination angles of two normal canine hearts were 50.6 and 46.2 degrees, while the angle of a failing canine heart was 57.4 degrees. This results well fit the anatomical knowledge that failing hearts tend to have vertical myocardical cell sheets.

  20. Obstructor-A is required for epithelial extracellular matrix dynamics, exoskeleton function, and tubulogenesis.

    PubMed

    Petkau, Georg; Wingen, Christian; Jussen, Laura C A; Radtke, Tina; Behr, Matthias

    2012-06-15

    The epidermis and internal tubular organs, such as gut and lungs, are exposed to a hostile environment. They form an extracellular matrix to provide epithelial integrity and to prevent contact with pathogens and toxins. In arthropods, the cuticle protects, shapes, and enables the functioning of organs. During development, cuticle matrix is shielded from premature degradation; however, underlying molecular mechanisms are poorly understood. Previously, we identified the conserved obstructor multigene-family, which encodes chitin-binding proteins. Here we show that Obstructor-A is required for extracellular matrix dynamics in cuticle forming organs. Loss of obstructor-A causes severe defects during cuticle molting, wound protection, tube expansion and larval growth control. We found that Obstructor-A interacts and forms a core complex with the polysaccharide chitin, the cuticle modifier Knickkopf and the chitin deacetylase Serpentine. Knickkopf protects chitin from chitinase-dependent degradation and deacetylase enzymes ensure extracellular matrix maturation. We provide evidence that Obstructor-A is required to control the presence of Knickkopf and Serpentine in the extracellular matrix. We propose a model suggesting that Obstructor-A coordinates the core complex for extracellular matrix protection from premature degradation. This mechanism enables exoskeletal molting, tube expansion, and epithelial integrity. The evolutionary conservation suggests a common role of Obstructor-A and homologs in coordinating extracellular matrix protection in epithelial tissues of chitinous invertebrates.

  1. The generic enhancement of photochromic dye switching speeds in a rigid polymer matrix.

    PubMed

    Evans, Richard A; Hanley, Tracey L; Skidmore, Melissa A; Davis, Thomas P; Such, Georgina K; Yee, Lachlan H; Ball, Graham E; Lewis, David A

    2005-03-01

    The switching or isomerization speed of photochromic dyes in a rigid polymeric matrix (such as an ophthalmic lens) is generally significantly slower than that observed in the mobile environment of a solution. Here we describe that the attachment of flexible oligomers having a low glass-transition temperature-such as poly(dimethylsiloxane)-to photochromic dyes greatly increases their switching speeds in a rigid polymer matrix. The greatest impact was observed in the thermal fade parameters T(1/2) and T(3/4)-the times it takes for the optical density to reduce by half and three quarters of the initial optical density of the coloured state-which were reduced by 40-95% and 60-99% respectively for spirooxazines, chromenes and an azo dye in a host polymer with a glass-transition temperature of 120 degrees C. The method does not alter the electronic nature of the dyes but simply protects them from the host matrix and provides greater molecular mobility for the switching process. In addition to ophthalmic lenses, the generic nature of the method may find further utility in data recording or optical switching.

  2. A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity.

    PubMed

    Liang, Youyun; Jeong, Jaehyun; DeVolder, Ross J; Cha, Chaenyung; Wang, Fei; Tong, Yen Wah; Kong, Hyunjoon

    2011-12-01

    Three dimensional (3D) tumor spheroid models are becoming important biomedical tools for both fundamental and applied cancer studies, but current models do not account for different levels of cancer malignancy. Several studies have reported that the mechanical rigidity of a hydrogel plays a significant role in regulating the phenotypes of cancer cells adhered to the gel surface. This finding suggests that matrix rigidity should also modulate the malignancy of 3D tumor spheroids. However, the role of matrix stiffness is often confounded by concurrent changes in 3D matrix permeability. This study reports an advanced strategy to assemble 3D liver tumor spheroids with controlled intercellular organization, phenotypes, and angiogenic activities using hydrogels with controlled stiffness and minimal differences in molecular diffusivity. The elastic moduli of cell-encapsulated collagen gels were increased by stiffening interconnected collagen fibers with varied amounts of poly(ethylene glycol) di-(succinic acid N-hydroxysuccinimidyl ester). Interestingly, hepatocellular carcinoma cells encapsulated in a fat-like, softer hydrogel formed malignant cancer spheroids, while cells cultured in a liver-like, stiffer gel formed compact hepatoids with suppressed malignancy. Overall, both the hydrogel and the 3D tumor spheroids developed in this study will be greatly useful to better understand and regulate the emergent behaviors of various cancer cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. A Chemomechanical Model of Matrix and Nuclear Rigidity Regulation of Focal Adhesion Size

    PubMed Central

    Cao, Xuan; Lin, Yuan; Driscoll, Tristian P.; Franco-Barraza, Janusz; Cukierman, Edna; Mauck, Robert L.; Shenoy, Vivek B.

    2015-01-01

    In this work, a chemomechanical model describing the growth dynamics of cell-matrix adhesion structures (i.e., focal adhesions (FAs)) is developed. We show that there are three regimes for FA evolution depending on their size. Specifically, nascent adhesions with initial lengths below a critical value that are yet to engage in actin fibers will dissolve, whereas bigger ones will grow into mature FAs with a steady state size. In adhesions where growth surpasses the steady state size, disassembly will occur until their sizes are reduced to the equilibrium state. This finding arises from the fact that polymerization of adhesion proteins is force-dependent. Under actomyosin contraction, individual integrin bonds within small FAs (i.e., nascent adhesions or focal complexes) must transmit higher loads while the phenomenon of stress concentration occurs at the edge of large adhesion patches. As such, an effective stiffness of the FA-extracellular matrix complex that is either too small or too large will be relatively low, resulting in a limited actomyosin pulling force developed at the edge that is insufficient to prevent disassembly. Furthermore, it is found that a stiffer extracellular matrix and/or nucleus, as well as a stronger chemomechanical feedback, will induce larger adhesions along with a higher level of contraction force. Interestingly, switching the extracellular side from an elastic half-space, corresponding to some widely used in vitro gel substrates, to a one-dimensional fiber (as in the case of cells anchoring to a fibrous scaffold in vivo) does not qualitative change these conclusions. Our model predictions are in good agreement with a variety of experimental observations obtained in this study as well as those reported in the literature. Furthermore, this new model, to our knowledge, provides a framework with which to understand how both intracellular and extracellular perturbations lead to changes in adhesion structure number and size. PMID:26536258

  4. Biological variation of extracellular matrix biomarkers in patients with stable chronic heart failure.

    PubMed

    Täger, Tobias; Wiebalck, Clara; Fröhlich, Hanna; Corletto, Anna; Katus, Hugo A; Frankenstein, Lutz

    2017-08-04

    Extracellular matrix (ECM) biomarkers such as matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are pathophysiological key, prognostic marker and therapeutic target in chronic heart failure (HF). Serial measurements of MMPs and TIMPs may be useful for guidance of these applications. However, interpretation of time-dependent changes requires knowledge about the biological variation of ECM biomarkers. We performed measurements of MMP-2, MMP-9, TIMP-1, and TIMP-4 in 50 patients with chronic HF who met rigid criteria for clinical stability at 3-h, 6-h, 1-week and 2-week time intervals. In addition, clinical and haemodynamic assessment was performed at baseline, at 1- and 2-week intervals. Haemodynamic variables were measured using inert gas rebreathing and impedance cardiography. Heart rhythm was monitored with external ECG event recorders throughout the complete study. Reference change values (RCVs) and minimal important differences (MIDs) were determined for MMP-2, MMP-9, TIMP-1, and TIMP-4. Clinical and haemodynamic variables were stable over time. Depending on the time-interval, RCVs ranged between 4.9 and 11.7% for MMP-2, 26.4 and 56.7% for MMP-9, 10.8 and 30.7% for TIMP-1, and 16.0 and 47.4% for TIMP-4, respectively. The MIDs varied between 43.38 and 65.22 ng/ml for MMP-2, 28.71 and 40.96 ng/ml for MMP-9, 52.32 and 156.07 ng/ml for TIMP-1, and 293.92 and 798.04 pg/ml for TIMP-4, respectively. The biological variation of ECM biomarkers differs with respect to individual biomarkers and time intervals. MMP-2 may be most suitable for serial biomarker measurements, as the biological variation is low irrespective of the time interval between measurements.

  5. Binding of Matrix Metalloproteinase Inhibitors to Extracellular Matrix: 3D-QSAR Analysis

    PubMed Central

    Zhang, Yufen; Lukacova, Viera; Bartus, Vladimir; Nie, Xiaoping; Sun, Guorong; Manivannan, Ethirajan; Ghorpade, Sandeep R.; Jin, Xiaomin; Manyem, Shankar; Sibi, Mukund P.; Cook, Gregory R.; Balaz, Stefan

    2008-01-01

    Binding to the extracellular matrix (ECM), one of the most abundant human protein complexes, significantly affects drug disposition. Specifically, the interactions with ECM determine the free concentrations of small molecules acting in tissues, including signaling peptides, inhibitors of tissue remodeling enzymes such as matrix metalloproteinases (MMPs), and other drug candidates. The nature of ECM binding was elucidated for 63 MMP inhibitors, for which the association constants to an ECM mimic were reported here. The data did not correlate with lipophilicity as a common determinant of structure-nonspecific, orientation-averaged binding. A hypothetical structure of the binding site of the solidified ECM surrogate was analyzed using the Comparative Molecular Field Analysis (CoMFA), which needed to be applied in our multi-mode variant. This fact indicates that the compounds bind to ECM in multiple modes, which cannot be considered as completely orientation-averaged and exhibit structural dependence. The novel CoMFA models, exhibiting satisfactory descriptive and predictive abilities, are suitable for prediction of the ECM binding for the untested chemicals, which are within applicability domains. The results contribute to a better prediction of the pharmacokinetic parameters such as the distribution volume and the tissue-blood partition coefficients, in addition to a more imminent benefit for the development of more effective MMP inhibitors. PMID:18844670

  6. Extracellular Matrix-Based Biohybrid Materials for Engineering Compliant, Matrix-Dense Tissues.

    PubMed

    Bracaglia, Laura G; Fisher, John P

    2015-11-18

    An ideal tissue engineering scaffold should not only promote, but take an active role in, constructive remodeling and formation of site appropriate tissue. Extracellular matrix (ECM)-derived proteins provide unmatched cellular recognition, and therefore influence cellular response towards predicted remodeling behaviors. Materials built with only these proteins, however, can degrade rapidly or begin too weak to substitute for compliant, matrix-dense tissues. The focus of this Progress Report is on biohybrid materials that incorporate polymer components with ECM-derived proteins, to produce a substrate with desired mechanical and degradation properties, as well as actively guide tissue remodeling. Materials are described through four fabrication methods: 1) polymer and ECM-protein fibers woven together, 2) polymer and ECM proteins combined in a bilayer, 3) cell-built ECM on polymer scaffold, and 4) ECM proteins and polymers combined in a single hydrogel. Scaffolds from each fabrication method can achieve characteristics suitable for different types of tissue. In vivo testing has shown progressive remodeling in injury models, and suggests ECM-based biohybrid materials promote a prohealing immune response over single component alternatives. The prohealing immune response is associated with lasting success and long term host maintenance of the implant. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Modifications in stromal extracellular matrix of aged corneas can be induced by ultraviolet A irradiation

    PubMed Central

    Gendron, Sébastien P; Rochette, Patrick J

    2015-01-01

    With age, structural and functional changes can be observed in human cornea. Some studies have shown a loss of corneal transparency and an increase in turbidity associated with aging. These changes are caused by modifications in the composition and arrangement of extracellular matrix in the corneal stroma. In human skin, it is well documented that exposure to solar radiation, and mainly to the UVA wavelengths, leads to phenotypes of photoaging characterized by alteration in extracellular matrix of the dermis. Although the cornea is also exposed to solar radiation, the extracellular matrix modifications observed in aging corneas have been mainly attributed to chronological aging and not to solar exposure. To ascertain the real implication of UVA exposure in extracellular matrix changes observed with age in human cornea, we have developed a model of photoaging by chronically exposing corneal stroma keratocytes with a precise UVA irradiation protocol. Using this model, we have analyzed UVA-induced transcriptomic and proteomic changes in corneal stroma. Our results show that cumulative UVA exposure causes changes in extracellular matrix that are found in corneal stromas of aged individuals, suggesting that solar exposure catalyzes corneal aging. Indeed, we observe a downregulation of collagen and proteoglycan gene expression and a reduction in proteoglycan production and secretion in response to cumulative UVA exposure. This study provides the first evidence that chronic ocular exposure to sunlight affects extracellular matrix composition and thus plays a role in corneal changes observed with age. PMID:25728164

  8. Altered protein levels in the isolated extracellular matrix of failing human hearts with dilated cardiomyopathy.

    PubMed

    DeAguero, Joshua L; McKown, Elizabeth N; Zhang, Liwen; Keirsey, Jeremy; Fischer, Edgar G; Samedi, Von G; Canan, Benjamin D; Kilic, Ahmet; Janssen, Paul M L; Delfín, Dawn A

    Dilated cardiomyopathy (DCM) is associated with extensive pathological cardiac remodeling and involves numerous changes in the protein expression profile of the extracellular matrix of the heart. We obtained seven human, end-stage, failing hearts with DCM (DCM-failing) and nine human, nonfailing donor hearts and compared their extracellular matrix protein profiles. We first showed that the DCM-failing hearts had indeed undergone extensive remodeling of the left ventricle myocardium relative to nonfailing hearts. We then isolated the extracellular matrix from a subset of these hearts and performed a proteomic analysis on the isolated matrices. We found that the levels of 26 structural proteins were altered in the DCM-failing isolated cardiac extracellular matrix compared to nonfailing isolated cardiac extracellular matrix. Overall, most of the extracellular matrix proteins showed reduced levels in the DCM-failing hearts, while all of the contractile proteins showed increased levels. There was a mixture of increased and decreased levels of cytoskeletal and nuclear transport proteins. Using immunoprobing, we verified that collagen IV (α2 and α6 isoforms), zyxin, and myomesin protein levels were reduced in the DCM-failing hearts. We expect that these data will add to the understanding of the pathology associated with heart failure with DCM.

  9. Cellular Force, and Geometry Sensing (Over Time) Can Detect Matrix Rigidity: Local Modules Produce Global Signals

    NASA Astrophysics Data System (ADS)

    Sheetz, Michael

    2006-03-01

    The shape and behavior of mammalian cells is defined by an interplay between extracellular signals and the cellular responses. Although the chemical nature of the external signals is important, there is a growing realization that the physical aspects of the external environment are equally important. In particular, the stresses, rigidity and form of the external environment have major effects on cell behavior. Of particular importance is rigidity since cancerous cells can often grow on soft agar or in a fluid phase without force production. For most mammalian cells there are relatively few types of motility that are evident from quantitative analyses of rapidly spreading fibroblasts (Dubin-Thaler et al., Biophys. J. 86:1794-1806, 2004). One motile phase that we have studied extensively involves periodic contractions (24 s period) in local regions of the leading edge of the cell (Giannone et al., Cell, 116:431-443, 2004). The periodic signal is carried radially from the cell edge toward the center and is part of a general mechanism for rigidity-directed movement and pathfinding. Another motile phase involves the movement of individual collagen fibers in a hand-over-hand fashion (Meshel et al., Nature Cell Biol. 7:157-164, 2005) where the form of the fiber is being sensed. Rigidity and form sensing in these systems is dependent upon the cytoskeleton and force-dependent tyrosine phosphorylation through oncogenes (Sawada and Sheetz, J Cell Biol. 156:609-15, 2002; Tamada et al., Developmental Cell, 7:706-718, 2004). Recent studies indicate that the cell rigidity sensing occurs preferentially at the leading edges of moving cells and involves forces of 10-20 pN generated by displacements of 50-100 nm (Jiang et al., Biophys J. In Press). We will discuss how cells organize motility tools in motile phases (Döbereiner et al., Phys. Rev. Letters. 93:108105-1-4, 2004) in a dialogue with the environment to define cell morphology and behavior over time..

  10. The bioactivity of cartilage extracellular matrix in articular cartilage regeneration.

    PubMed

    Sutherland, Amanda J; Converse, Gabriel L; Hopkins, Richard A; Detamore, Michael S

    2015-01-07

    Cartilage matrix is a promising material for cartilage regeneration given the evidence supporting its chondroinductive character. The "raw materials" of cartilage matrix can serve as building blocks and signals for tissue regeneration. These matrices can be created by chemical or physical processing: physical methods disrupt cellular membranes and nuclei but may not fully remove all cell components and DNA, whereas chemical methods combined with physical methods are effective in fully decellularizing such materials. It is important to delineate between the sources of the cartilage matrix, that is, derived from matrix in vitro or from native tissue, and then to further characterize the cartilage matrix based on the processing method, decellularization or devitalization. With these distinctions, four types of cartilage matrices exist: decellularized native cartilage (DCC), devitalized native cartilage (DVC), decellularized cell-derived matrix (DCCM), and devitalized cell-derived matrix (DVCM). One currently marketed cartilage matrix device is decellularized, although trends in patents suggest additional decellularized products may be available in the future. To identify the most relevant source and processing for cartilage matrix, testing needs to include targeting the desired application, optimizing delivery of the material, identify relevant FDA regulations, assess availability of materials, and immunogenic properties of the product.

  11. The extracellular matrix: Structure, composition, age-related differences, tools for analysis and applications for tissue engineering.

    PubMed

    Kular, Jaspreet K; Basu, Shouvik; Sharma, Ram I

    2014-01-01

    The extracellular matrix is a structural support network made up of diverse proteins, sugars and other components. It influences a wide number of cellular processes including migration, wound healing and differentiation, all of which is of particular interest to researchers in the field of tissue engineering. Understanding the composition and structure of the extracellular matrix will aid in exploring the ways the extracellular matrix can be utilised in tissue engineering applications especially as a scaffold. This review summarises the current knowledge of the composition, structure and functions of the extracellular matrix and introduces the effect of ageing on extracellular matrix remodelling and its contribution to cellular functions. Additionally, the current analytical technologies to study the extracellular matrix and extracellular matrix-related cellular processes are also reviewed.

  12. SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

    PubMed

    Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene

    2005-10-28

    SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

  13. Extracellular Matrix Components Regulate Cellular Polarity and Tissue Structure in the Developing and Mature Retina.

    PubMed

    Varshney, Shweta; Hunter, Dale D; Brunken, William J

    2015-01-01

    While genetic networks and other intrinsic mechanisms regulate much of retinal development, interactions with the extracellular environment shape these networks and modify their output. The present review has focused on the role of one family of extracellular matrix molecules and their signaling pathways in retinal development. In addition to their effects on the developing retina, laminins play a role in maintaining Müller cell polarity and compartmentalization, thereby contributing to retinal homeostasis. This article which is intended for the clinical audience, reviews the fundamentals of retinal development, extracellular matrix organization and the role of laminins in retinal development. The role of laminin in cortical development is also briefly discussed.

  14. The Bioactivity of Cartilage Extracellular Matrix in Articular Cartilage Regeneration

    PubMed Central

    Sutherland, Amanda J.; Converse, Gabriel L.; Hopkins, Richard A.; Detamore, Michael S.

    2014-01-01

    Cartilage matrix is a particularly promising acellular material for cartilage regeneration given the evidence supporting its chondroinductive character. The ‘raw materials’ of cartilage matrix can serve as building blocks and signals for enhanced tissue regeneration. These matrices can be created by chemical or physical methods: physical methods disrupt cellular membranes and nuclei but may not fully remove all cell components and DNA, whereas chemical methods when combined with physical methods are particularly effective in fully decellularizing such materials. Critical endpoints include no detectable residual DNA or immunogenic antigens. It is important to first delineate between the sources of the cartilage matrix, i.e., derived from matrix produced by cells in vitro or from native tissue, and then to further characterize the cartilage matrix based on the processing method, i.e., decellularization or devitalization. With these distinctions, four types of cartilage matrices exist: decellularized native cartilage (DCC), devitalized native cartilage (DVC), decellularized cell derived matrix (DCCM), and devitalized cell derived matrix (DVCM). Delivery of cartilage matrix may be a straightforward approach without the need for additional cells or growth factors. Without additional biological additives, cartilage matrix may be attractive from a regulatory and commercialization standpoint. Source and delivery method are important considerations for clinical translation. Only one currently marketed cartilage matrix medical device is decellularized, although trends in filed patents suggest additional decellularized products may be available in the future. To choose the most relevant source and processing for cartilage matrix, qualifying testing needs to include targeting the desired application, optimizing delivery of the material, identify relevant FDA regulations, assess availability of raw materials, and immunogenic properties of the product. PMID:25044502

  15. Traction Force Microscopy in 3-Dimensional Extracellular Matrix Networks.

    PubMed

    Cóndor, M; Steinwachs, J; Mark, C; García-Aznar, J M; Fabry, B

    2017-06-19

    Cell migration through a three-dimensional (3-D) matrix depends strongly on the ability of cells to generate traction forces. To overcome the steric hindrance of the matrix, cells need to generate sufficiently high traction forces but also need to distribute these forces spatially in a migration-promoting way. This unit describes a protocol to measure spatial maps of cell traction forces in 3-D biopolymer networks such as collagen, fibrin, or Matrigel. Traction forces are computed from the relationship between measured force-induced matrix deformations surrounding the cell and the known mechanical properties of the matrix. The method does not rely on knowledge of the cell surface coordinates and takes nonlinear mechanical properties of the matrix into account. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  16. The tetrapartite synapse: Extracellular matrix remodeling contributes to corticoaccumbens plasticity underlying drug addiction.

    PubMed

    Smith, Alexander C W; Scofield, Michael D; Kalivas, Peter W

    2015-12-02

    Synaptic plasticity has long been known to involve three key elements of neuropil, the presynapse, the postsynapse and adjacent glia. Here we review the role of the extracellular matrix in synaptic plasticity as a necessary component forming the tetrapartite synapse. We describe the role of matrix metalloproteinases as enzymes sculpting extracellular proteins and thereby creating an extracellular signaling domain required for synaptic plasticity. Specifically we focus on the role of the tetrapartite synapse in mediating the effects of addictive drugs at cortico-striatal synapses, and conclude that the extracellular signaling domain and its regulation by matrix metalloproteinases is critical for developing and expressing drug seeking behaviors. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Molecular Control of Vascular Tube Morphogenesis and Stabilization: Regulation by Extracellular Matrix, Matrix Metalloproteinases, and Endothelial Cell-Pericyte Interactions

    NASA Astrophysics Data System (ADS)

    Davis, George E.; Stratman, Amber N.; Sacharidou, Anastasia

    Recent studies have revealed a critical role for both extracellular matrices and matrix metalloproteinases in the molecular control of vascular morphogenesis and stabilization in three-dimensional (3D) tissue environments. Key interactions involve endothelial cells (ECs) and pericytes, which coassemble to affect vessel formation, remodeling, and stabilization events during development and postnatal life. EC-pericyte interactions control extracellular matrix remodeling events including vascular basement membrane matrix assembly, a necessary step for endothelial tube maturation and stabilization. ECs form tube networks in 3D extracellular matrices in a manner dependent on integrins, membrane-type metalloproteinases, and the Rho GTPases, Cdc42 and Rac1. Recent work has defined an EC lumen signaling complex of proteins composed of these proteins that controls 3D matrix-specific signaling events required for these processes. The EC tube formation process results in the creation of a network of proteolytically generated vascular guidance tunnels. These tunnels are physical matrix spaces that regulate vascular tube remodeling and represent matrix conduits into which pericytes are recruited to allow dynamic cell-cell interactions with ECs. These dynamic EC-pericyte interactions induce vascular basement membrane matrix deposition, leading to vessel maturation and stabilization.

  18. The extracellular matrix and blood vessel formation: not just a scaffold

    PubMed Central

    Rhodes, John M; Simons, Michael

    2007-01-01

    Abstract The extracellular matrix plays a number of important roles, among them providing structural support and information to cellular structures such as blood vessels imbedded within it. As more complex organisms have evolved, the matrix ability to direct signalling towards the vasculature and remodel in response to signalling from the vasculature has assumed progressively greater importance. This review will focus on the molecules of the extracellular matrix, specifically relating to vessel formation and their ability to signal to the surrounding cells to initiate or terminate processes involved in blood vessel formation. PMID:17488472

  19. Angiogenic Type I Collagen Extracellular Matrix Integrated with Recombinant Bacteriophages Displaying Vascular Endothelial Growth Factors.

    PubMed

    Yoon, Junghyo; Korkmaz Zirpel, Nuriye; Park, Hyun-Ji; Han, Sewoon; Hwang, Kyung Hoon; Shin, Jisoo; Cho, Seung-Woo; Nam, Chang-Hoon; Chung, Seok

    2016-01-21

    Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

    PubMed

    Bush, Katie A; Downing, Brett R; Walsh, Sarah E; Pins, George D

    2007-02-01

    The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

  1. Anisotropic properties of the enamel organic extracellular matrix.

    PubMed

    do Espírito Santo, Alexandre R; Novaes, Pedro D; Line, Sérgio R P

    2006-05-01

    Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. This supramolecular ensemble controls the nucleation of the crystalline mineral phase. The detection of anisotropic properties by polarizing microscopy has been extensively used to detect macromolecular organizations in ordinary histological sections. The aim of this work was to study the birefringence of enamel organic matrix during the development of rat molar and incisor teeth. Incisor and molar teeth of rats were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2, and decalcified in 5% nitric acid/4% formaldehyde. After paraffin embedding, 5-microm-thick sections were obtained, treated with xylene, and hydrated. Form birefringence curves were obtained after measuring optical retardations in imbibing media, with different refractive indices. Our observations showed that enamel organic matrix of rat incisor and molar teeth is strongly birefringent, presenting an ordered supramolecular structure. The birefringence starts during the early secretion phase and disappears at the maturation phase. The analysis of enamel organic matrix birefringence may be used to detect the effects of genetic and environmental factors on the supramolecular orientation of enamel matrix and their effects on the structure of mature enamel.

  2. Detection of extracellular matrix modification in cancer models with inverse spectroscopic optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Spicer, Graham L. C.; Azarin, Samira M.; Yi, Ji; Young, Scott T.; Ellis, Ronald; Bauer, Greta M.; Shea, Lonnie D.; Backman, Vadim

    2016-10-01

    In cancer biology, there has been a recent effort to understand tumor formation in the context of the tissue microenvironment. In particular, recent progress has explored the mechanisms behind how changes in the cell-extracellular matrix ensemble influence progression of the disease. The extensive use of in vitro tissue culture models in simulant matrix has proven effective at studying such interactions, but modalities for non-invasively quantifying aspects of these systems are scant. We present the novel application of an imaging technique, Inverse Spectroscopic Optical Coherence Tomography, for the non-destructive measurement of in vitro biological samples during matrix remodeling. Our findings indicate that the nanoscale-sensitive mass density correlation shape factor D of cancer cells increases in response to a more crosslinked matrix. We present a facile technique for the non-invasive, quantitative study of the micro- and nano-scale structure of the extracellular matrix and its host cells.

  3. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases

    PubMed Central

    1995-01-01

    Interaction of cells with extracellular matrix via integrin adhesion receptors plays an important role in a wide range of cellular: functions, for example cell growth, movement, and differentiation. Upon interaction with substrate, integrins cluster and associate with a variety of cytoplasmic proteins to form focal complexes and with the actin cytoskeleton. Although the intracellular signals induced by integrins are at present undefined, it is thought that they are mediated by proteins recruited to the focal complexes. It has been suggested, for example, that after recruitment to focal adhesions p125FAK can activate the ERK1/2 MAP kinase cascade. We have previously reported that members of the rho family of small GTPases can trigger the assembly of focal complexes when activated in cells. Using microinjection techniques, we have now examined the role of the extracellular matrix and of the two GTP-binding proteins, rac and rho, in the assembly of integrin complexes in both mouse and human fibroblasts. We find that the interaction of integrins with extracellular matrix alone is not sufficient to induce integrin clustering and focal complex formation. Similarly, activation of rho or rac by extracellular growth factors does not lead to focal complex formation in the absence of matrix. Focal complexes are only assembled in the presence of both matrix and functionally active members of the rho family. In agreement with this, the interaction of integrins with matrix in the absence of rho/rac activity is unable to activate the ERK1/2 kinases in Swiss 3T3 cells. In fact, ERK1/2 can be activated fully by growth factors in the absence of matrix and it seems unlikely, therefore, that the adhesion dependence of fibroblast growth is mediated through the ras/MAP kinase pathway. We conclude that extracellular matrix is not sufficient to trigger focal complex assembly and subsequent integrin-dependent signal transduction in the absence of functionally active members of the rho

  4. Engineering 3D bio-artificial heart muscle: the acellular ventricular extracellular matrix model.

    PubMed

    Patel, Nikita M; Tao, Ze-Wei; Mohamed, Mohamed A; Hogan, Matt K; Gutierrez, Laura; Birla, Ravi K

    2015-01-01

    Current therapies in left ventricular systolic dysfunction and end-stage heart failure include mechanical assist devices or transplant. The development of a tissue-engineered integrative platform would present a therapeutic option that overcomes the limitations associated with current treatment modalities. This study provides a foundation for the fabrication and preliminary viability of the acellular ventricular extracellular matrix (AVEM) model. Acellular ventricular extracellular matrix was fabricated by culturing 4 million rat neonatal cardiac cells around an excised acellular ventricular segment. Acellular ventricular extracellular matrix generated a maximum spontaneous contractile force of 388.3 μN and demonstrated a Frank-Starling relationship at varying pretensions. Histologic assessment displayed cell cohesion and adhesion within the AVEM as a result of passive cell seeding.

  5. Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

    SciTech Connect

    Hansen, R K; Bissell, M J

    2000-06-01

    The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function.

  6. Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

    PubMed Central

    Hansen, R K; Bissell, M J

    2010-01-01

    The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function. PMID:10903527

  7. Symposium: Role of the extracellular matrix in mammary development. Regulation of milk protein and basement membrane gene expression: The influence of the extracellular matrix

    SciTech Connect

    Aggeler, J.; Park, C.S.; Bissell, M.J.

    1988-10-01

    Synthesis and secretion of milk proteins ({alpha}-casein, {beta}-casein, {gamma}-casein, and transferrin) by cultured primary mouse mammary epithelial cells is modulated by the extracellular matrix. In cells grown on released or floating type I collagen gels, mRNA for {beta}-casein and transferrin is increased as much as 30-fold over cells grown on plastic. Induction of {beta}-casein expression depends strongly on the presence of lactogenic hormones, especially prolactin, in the culture. When cells are plated onto partially purified reconstituted basement membrane, dramatic changes in morphology and milk protein gene expression are observed. Cells cultured on the matrix for 6 to 8 d in the presence of prolactin, insulin, and hydrocortisone form hollow spheres and duct-like structures that are completely surrounded by matrix. The cells lining these spheres appear actively secretory and are oriented with their apices facing the lumen. Hybridization experiments indicate that mRNA for {beta}-casein can be increased as much as 70-fold in these cultures. Because > 90% of the cultured cells synthesize immunoreactive {beta}-casein, as compared with only 40% of cells in the late pregnant gland, the matrix appears to be able to induce protein expression in previously silent cells. Synthesis of laminin and assembly of a mammary-specific basal lamina by cells cultured on different extracellular matrices also appears to depend on the presence of lactogenic hormones. These studies provide support for the concept of dynamic reciprocity in which complex interactions between extracellular matrix and the cellular cytoskeleton contribute to the induction and maintenance of tissue-specific gene expression in the mammary gland.

  8. Depressed immune surveillance against cancer: role of deficient T cell: extracellular matrix interactions.

    PubMed

    Górski, A; Castronovo, V; Stepień-Sopniewska, B; Grieb, P; Ryba, M; Mrowiec, T; Korczak-Kowalska, G; Wierzbicki, P; Matysiak, W; Dybowska, B

    1994-07-01

    Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.

  9. An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans.

    PubMed

    Byrd, Angel S; O'Brien, Xian M; Johnson, Courtney M; Lavigne, Liz M; Reichner, Jonathan S

    2013-04-15

    The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as neutrophil extracellular traps (NETs). The receptor/ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Because the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrix, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern β-glucan by human neutrophils causes rapid (≤ 30 min) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized β-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn with β-glucan is robust, accounting for 17.2 ± 3.4% of total DNA in the cell population. Release is dependent on β-glucan recognition by complement receptor 3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on β-glucan recognition by complement receptor 3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid antifungal response.

  10. Alterations in the biosynthesis of extracellular matrix molecules in connective tissues by electric and magnetic fields

    SciTech Connect

    Ciombor, D.M.

    1992-01-01

    Pulsed electromagnetic fields (PEMFs) of certain configurations have been shown to be effective clinically in promoting the healing of fracture non-unions and are believed to enhance calcification of extracellular matrix. In vitro studies have suggested that PEMFs may also have the effect of modifying the extracellular matrix by promoting the synthesis of matrix molecules. This study examines the effect of one particular type of PEMF and a sinusoidal continuous wave upon the extracellular matrix and calcification of endochondral ossification in vivo. The pulsed magnetic field (SS-22) utilized in these studies is being used clinically for the treatment of fracture non-unions, a condition in which the bone is not restored to form or function. The sinusoidal continuous wave was designed to provide a 5 Gauss amplitude at a 15 Hz. rate. The synthesis of cartilage molecules is enhanced by this type of PEMF and since wave and subsequent endochondral calcification is stimulated. Histomorphometric studies indicate that the maturation of bone trabeculae is also promoted by this type of PEMF stimulation. These results indicate that a specific PEMF or continuous waveform can change the composition of cartilage extracellular matrix in vivo and raises the possibility that the effects on other processes of endochondral ossification (e.g., fracture healing and growth plates) may occur through a similar mechanism.

  11. Topological Control of Extracellular Matrix Growth: A Native-Like Model for Cell Morphodynamics Studies.

    PubMed

    Caballero, David; Samitier, Josep

    2017-02-01

    The interaction of cells with their natural environment influences a large variety of cellular phenomena, including cell adhesion, proliferation, and migration. The complex extracellular matrix network has challenged the attempts to replicate in vitro the heterogeneity of the cell environment and has threatened, in general, the relevance of in vitro studies. In this work, we describe a new and extremely versatile approach to generate native-like extracellular matrices with controlled morphologies for the in vitro study of cellular processes. This general approach combines the confluent culture of fibroblasts with microfabricated guiding templates to direct the three-dimensional growth of well-defined extracellular networks which recapitulate the structural and biomolecular complexity of features typically found in vivo. To evaluate its performance, we studied fundamental cellular processes, including cell cytoskeleton organization, cell-matrix adhesion, proliferation, and protrusions morphodynamics. In all cases, we found striking differences depending on matrix architecture and, in particular, when compared to standard two-dimensional environments. We also assessed whether the engineered matrix networks influenced cell migration dynamics and locomotion strategy, finding enhanced migration efficiency for cells seeded on aligned matrices. Altogether, our methodology paves the way to the development of high-performance models of the extracellular matrix for potential applications in tissue engineering, diagnosis, or stem-cell biology.

  12. Mammary epithelial cell: Influence of extracellular matrix composition and organization during development and tumorigenesis

    PubMed Central

    Kass, Laura; Erler, Janine T.; Dembo, Micah; Weaver, Valerie M.

    2009-01-01

    Stromal–epithelial interactions regulate mammary gland development and are critical for the maintenance of tissue homeostasis. The extracellular matrix, which is a proteinaceous component of the stroma, regulates mammary epithelial growth, survival, migration and differentiation through a repertoire of transmembrane receptors, of which integrins are the best characterized. Integrins modulate cell fate by reciprocally transducing biochemical and biophysical cues between the cell and the extracellular matrix, facilitating processes such as embryonic branching morphogenesis and lactation in the mammary gland. During breast development and cancer progression, the extracellular matrix is dynamically altered such that its composition, turnover, processing and orientation change dramatically. These modifications influence mammary epithelial cell shape, and modulate growth factor and hormonal responses to regulate processes including branching morphogenesis and alveolar differentiation. Malignant transformation of the breast is also associated with significant matrix remodeling and a progressive stiffening of the stroma that can enhance mammary epithelial cell growth, perturb breast tissue organization, and promote cell invasion and survival. In this review, we discuss the role of stromal–epithelial interactions in normal and malignant mammary epithelial cell behavior. We specifically focus on how dynamic modulation of the biochemical and biophysical properties of the extracellular matrix elicit a dialogue with the mammary epithelium through transmembrane integrin receptors to influence tissue morphogenesis, homeostasis and malignant transformation. PMID:17719831

  13. Extracellular-matrix tethering regulates stem-cell fate

    NASA Astrophysics Data System (ADS)

    Trappmann, Britta; Gautrot, Julien E.; Connelly, John T.; Strange, Daniel G. T.; Li, Yuan; Oyen, Michelle L.; Cohen Stuart, Martien A.; Boehm, Heike; Li, Bojun; Vogel, Viola; Spatz, Joachim P.; Watt, Fiona M.; Huck, Wilhelm T. S.

    2012-07-01

    To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

  14. Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

    PubMed Central

    Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

    2014-01-01

    The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method. PMID:24887553

  15. Cooperation of Ito cells and hepatocytes in the deposition of an extracellular matrix in vitro.

    PubMed Central

    Loréal, O.; Levavasseur, F.; Fromaget, C.; Gros, D.; Guillouzo, A.; Clément, B.

    1993-01-01

    Cellular and molecular mechanisms involved in the deposition of extracellular matrix components in both normal and fibrotic liver are still poorly understood. We have investigated the influence of cooperation between Ito cells and hepatocytes in matrix deposition in vitro. Immunoprecipitation of radiolabeled proteins from media of 5-day-old Ito cell primary cultures showed that these cells secreted high levels of the major basement membrane components, ie, collagen IV, laminin, and entactin/nidogen. By immunocytochemistry, precursors of basement membrane components were found intracellularly, but only scarce deposits were seen around the cells. When hepatocytes were added to 2-day-old Ito cell primary cultures, they established close contacts with Ito cells in less than 24 hours and expressed ZO-1, a tight junction-associated protein not detectable in standard hepatocyte culture. Cytochemistry analysis revealed an abundant extracellular matrix deposited over hepatocyte cords and between hepatocytes and Ito cells. Immunocytochemistry studies showed that this matrix contained laminin, fibronectin, and collagens proIII and IV. These data indicate that a high level of matrix protein synthesis by liver cells in vitro is not sufficient to induce extracellular matrix deposition, and that cell-cell interactions are strongly involved in this process. Hepatocyte/Ito cell co-culture, which may reflect the actual situation in vivo, represents a useful tool for studying liver fibrogenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8342601

  16. Physicomechanical properties of the extracellular matrix of a demineralized bone

    NASA Astrophysics Data System (ADS)

    Kirilova, I. A.; Sharkeev, Yu. P.; Nikolaev, S. V.; Podorozhnaya, V. T.; Uvarkin, P. V.; Ratushnyak, A. S.; Chebodaeva, V. V.

    2016-08-01

    The article describes the results of a study of physicomechanical properties of a demineralized bone matrix of human cancellous and compact bones. A demineralized cancellous bone was shown to have the best characteristics of a porous system for colonization of matrices by cells. The ultimate stress and elasticity modulus of samples of demineralized femoral heads isolated in primary hip replacement was demonstrated to vary in wide ranges. The elasticity modulus ranged from 50 to 250 MPa, and the tensile strength varied from 1.1 to 5.5 MPa. Microhardness measurements by the recovered indentation method were not possible because of the viscoelastic properties of a bone material. To study the piezoelectric properties of samples, a measuring system was developed that comprised a measuring chamber with contact electrodes, a system for controlled sample loading, an amplifier-converter unit, and signal recording and processing software. The measurement results were used to determine the dependence of the signal amplitude on the dynamic deformation characteristics. The findings are discussed in terms of the relationship between the mechanical and electrical properties and the structure of the organic bone component.

  17. Bicomponent electrospun scaffolds to design extracellular matrix tissue analogs.

    PubMed

    Guarino, Vincenzo; Cirillo, Valentina; Ambrosio, Luigi

    2016-01-01

    In the last decade, bicomponent fibers have been proposed to fabricate bio-inspired systems for tissue repair, regenerative medicine, medical healthcare and clinical applications. In comparison with monocomponent fibers, key advantage concerns their ability of self-adapting to the physiological conditions through an extended pattern of signals--morphological, chemical and physical ones--confined at the single fiber level. Hydrophobic/hydrophilic phases may be variously organized by tuneable processing modes (i.e., blending, core/shell, interweaving) thus offering different benefits in terms of biological activity, fluid sorption and molecular transport properties (first generation). The possibility to efficiently graft cell-adhesive proteins and peptide sequences onto the fiber surface mediated by spacers or impregnating hydrogels allows to trigger cell late activities by a controlled and sustained release in vitro of specific biomolecules (i.e., morphogens, growth factors). Here, we introduce an overview of current approaches based on bicomponent fiber use as extra cellular matrix analogs with cell-instructive functions and hierarchal organization of living tissues.

  18. A Hydrogel Derived From Decellularized Dermal Extracellular Matrix

    PubMed Central

    Wolf, Matthew T.; Daly, Kerry A.; Brennan-Pierce, Ellen P.; Johnson, Scott A.; Carruthers, Christopher; D’Amore, Antonio; Nagarkar, Shailesh P.; Velankar, Sachin S.; Badylak, Stephen F.

    2012-01-01

    The ECM of mammalian tissues has been used as a scaffold to facilitate the repair and reconstruction of numerous tissues. Such scaffolds are prepared in many forms including sheets, powders, and hydrogels. ECM hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the inherent bioactivity of native matrix. However, material properties of ECM hydrogels and the effect of these properties upon cell behavior are neither well understood nor controlled. The objective of this study was to prepare and determine the structure, mechanics, and the cell response in vitro and in vivo of ECM hydrogels prepared from decellularized porcine dermis and urinary bladder tissues. Dermal ECM hydrogels were characterized by a more dense fiber architecture and greater mechanical integrity than urinary bladder ECM hydrogels, and showed a dose dependent increase in mechanical properties with ECM concentration. In vitro, dermal ECM hydrogels supported greater C2C12 myoblast fusion, and less fibroblast infiltration and less fibroblast mediated hydrogel contraction than urinary bladder ECM hydrogels. Both hydrogels were rapidly infiltrated by host cells, primarily macrophages, when implanted in a rat abdominal wall defect. Both ECM hydrogels degraded by 35 days in vivo, but UBM hydrogels degraded more quickly, and with greater amounts of myogenesis than dermal ECM. These results show that ECM hydrogel properties can be varied and partially controlled by the scaffold tissue source, and that these properties can markedly affect cell behavior. PMID:22789723

  19. Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution

    PubMed Central

    Baiocchini, Andrea; Montaldo, Claudia; Conigliaro, Alice; Grimaldi, Alessio; Correani, Virginia; Mura, Francesco; Ciccosanti, Fabiola; Rotiroti, Nicolina; Brenna, Alessia; Montalbano, Marzia; D’Offizi, Gianpiero; Capobianchi, Maria Rosaria; Alessandro, Riccardo; Piacentini, Mauro; Schininà, Maria Eugenia; Maras, Bruno; Del Nonno, Franca; Tripodi, Marco; Mancone, Carmine

    2016-01-01

    Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies. PMID:26998606

  20. Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

    PubMed Central

    Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J.; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I.

    2015-01-01

    Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro. PMID:25736020

  1. Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior.

    PubMed

    Zeiger, Adam S; Loe, Felicia C; Li, Ran; Raghunath, Michael; Van Vliet, Krystyn J

    2012-01-01

    Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.

  2. Multiphase modelling of tumour growth and extracellular matrix interaction: mathematical tools and applications.

    PubMed

    Preziosi, Luigi; Tosin, Andrea

    2009-04-01

    Resorting to a multiphase modelling framework, tumours are described here as a mixture of tumour and host cells within a porous structure constituted by a remodelling extracellular matrix (ECM), which is wet by a physiological extracellular fluid. The model presented in this article focuses mainly on the description of mechanical interactions of the growing tumour with the host tissue, their influence on tumour growth, and the attachment/detachment mechanisms between cells and ECM. Starting from some recent experimental evidences, we propose to describe the interaction forces involving the extracellular matrix via some concepts coming from viscoplasticity. We then apply the model to the description of the growth of tumour cords and the formation of fibrosis.

  3. Novel extracellular matrix and microtubule cables associated with pseudopodia of Astrammina rara, a carnivorous Antarctic foraminifer.

    PubMed

    Bowser, S S; DeLaca, T E; Rieder, C L

    1986-02-01

    Astrammina rara is a benthic foraminiferan protozoan which uses an extensive network of fine, branching, and anastomosing pseudopodia to capture and digest metazoans up to 12 mm long. During such predation the pseudopodia appear remarkably elastic and tensile. Electron microscopy has revealed a novel extracellular matrix of thin branching fibers associated with A. rara's pseudopodia, which are otherwise typical in appearance. These fibers are structurally associated with the pseudopodial glycocalyx. Cytoplasmic microtubules are often found in close juxtaposition to the plasma membrane and overlying extracellular fibers. In the main pseudopodial trunks bundles of 30-300 microtubules are surrounded by a lightly staining matrix and linked by occasional bridges. The microtubules follow straight trajectories in distal filopodia, but in pseudopodial trunks they appear to coil around one another to form cables. These microtubular cables and extracellular fibers are novel features of A. rara's pseudopodia and may provide the structural basis for their tensile strength.

  4. Elastic fiber formation: a dynamic view of extracellular matrix assembly using timer reporters.

    PubMed

    Kozel, Beth A; Rongish, Brenda J; Czirok, Andras; Zach, Julia; Little, Charles D; Davis, Elaine C; Knutsen, Russell H; Wagenseil, Jessica E; Levy, Marilyn A; Mecham, Robert P

    2006-04-01

    To study the dynamics of elastic fiber assembly, mammalian cells were transfected with a cDNA construct encoding bovine tropoelastin in frame with the Timer reporter. Timer is a derivative of the DsRed fluorescent protein that changes from green to red over time and, hence, can be used to distinguish new from old elastin. Using dynamic imaging microscopy, we found that the first step in elastic fiber formation is the appearance of small cell surface-associated elastin globules that increased in size with time (microassembly). The elastin globules are eventually transferred to pre-existing elastic fibers in the extracellular matrix where they coalesce into larger structures (macroassembly). Mechanical forces associated with cell movement help shape the forming, extracellular elastic fiber network. Time-lapse imaging combined with the use of Timer constructs provides unique tools for studying the temporal and spatial aspects of extracellular matrix formation by live cells.

  5. Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior

    PubMed Central

    Zeiger, Adam S.; Loe, Felicia C.; Li, Ran; Raghunath, Michael; Van Vliet, Krystyn J.

    2012-01-01

    Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro. PMID:22649562

  6. First demonstration of decorin, an extracellular matrix molecule, in bovine mammary tissue

    USDA-ARS?s Scientific Manuscript database

    In the mammary gland, the extracellular matrix (ECM) is secreted by and surrounds cells located in both mammary parenchyma (PAR) and stroma. Decorin is an ECM proteoglycan with cell growth regulatory effects mediated by its ability to interact with growth factors or up-regulation of cyclin-dependent...

  7. Extracellular Matrix Formation Enhances the Ability of Streptococcus pneumoniae to Cause Invasive Disease

    PubMed Central

    Trappetti, Claudia; Ogunniyi, Abiodun D.; Oggioni, Marco R.; Paton, James C.

    2011-01-01

    During infection, pneumococci exist mainly in sessile biofilms rather than in planktonic form, except during sepsis. However, relatively little is known about how biofilms contribute to pneumococcal pathogenesis. Here, we carried out a biofilm assay on opaque and transparent variants of a clinical serotype 19F strain WCH159. After 4 days incubation, scanning electron microscopy revealed that opaque biofilm bacteria produced an extracellular matrix, whereas the transparent variant did not. The opaque biofilm-derived bacteria translocated from the nasopharynx to the lungs and brain of mice, and showed 100-fold greater in vitro adherence to A549 cells than transparent bacteria. Microarray analysis of planktonic and sessile bacteria from transparent and opaque variants showed differential gene expression in two operons: the lic operon, which is involved in choline uptake, and in the two-component system, ciaRH. Mutants of these genes did not form an extracellular matrix, could not translocate from the nasopharynx to the lungs or the brain, and adhered poorly to A549 cells. We conclude that only the opaque phenotype is able to form extracellular matrix, and that the lic operon and ciaRH contribute to this process. We propose that during infection, extracellular matrix formation enhances the ability of pneumococci to cause invasive disease. PMID:21611130

  8. Marrow stromal fibroblastic cell cultivation in vitro on decellularized bone marrow extracellular matrix.

    PubMed

    Dutra, Timothy F; French, Samuel W

    2010-02-01

    The in vitro biocompatibility of decellularized bone marrow extracellular matrix was evaluated. Following a freeze-thaw cycle, sectioned discs of fresh frozen rat metaphyseal bone were sequentially incubated in solutions of hypertonic, then hypotonic Ringer's solution, followed by deoxycholic acid, then DNAase I. The adequacy of decellularization of marrow stroma was examined by light microscopy. Marrow stromal fibroblastic cells were harvested by dispersion of rat long bone marrow, followed by concentration by discontinuous Ficoll-Paque gradient centrifugation. The fibroblastic cells were expanded by in vitro cultivation, and second passage cells were cryopreserved until needed. Cryopreserved marrow stromal cells were applied dropwise to sections of decellularized bone marrow extracellular matrix, and cultured in BJGb medium with 20% fetal bovine serum for ten days. Mature cultures were formalin fixed, decalcified, and embedded in paraffin. Light microscopy of hematoxylin and eosin stained sections showed individual spindle cells invading the upper portion of the decellularized extracellular matrix, and also a monolayer of spindle cells on the upper surfaces of exposed trabecular and cortical bone. This experiment showed that decellularized marrow extracellular matrix is a biocompatible three dimensional in vitro substrate for marrow stromal fibroblastic cells.

  9. Local fluid transfer regulation in heart extracellular matrix.

    PubMed

    McGee, Maria P; Morykwas, Michael J; Jordan, James E; Wang, Rui; Argenta, Louis C

    2016-06-01

    The interstitial myocardial matrix is a complex and dynamic structure that adapts to local fluctuations in pressure and actively contributes to the heart's fluid exchange and hydration. However, classical physiologic models tend to treat it as a passive conduit for water and solute, perhaps because local interstitial regulatory mechanisms are not easily accessible to experiment in vivo. Here, we examined the interstitial contribution to the fluid-driving pressure ex vivo. Interstitial hydration potentials were determined from influx/efflux rates measured in explants from healthy and ischemia-reperfusion-injured pigs during colloid osmotic pressure titrations. Adaptive responses were further explored by isolating myocardial fibroblasts and measuring their contractile responses to water activity changes in vitro. Results show hydration potentials between 5 and 60 mmHg in healthy myocardia and shifts in excess of 200 mmHg in edematous myocardia after ischemia-reperfusion injury. Further, rates of fluid transfer were temperature-dependent, and in collagen gel contraction assays, myocardial fibroblasts tended to preserve the micro-environment's hydration volume by slowing fluid efflux rates at pressures above 40 mmHg. Our studies quantify components of the fluid-driving forces in the heart interstitium that the classical Starling's equation does not explicitly consider. Measured hydration potentials in healthy myocardia and shifts with edema are larger than predicted from the known values of hydrostatic and colloid osmotic interstitial fluid pressures. Together with fibroblast responses in vitro, they are consistent with regulatory mechanisms that add local biological controls to classic fluid-balance models.

  10. Morphological Characterization of Organized Extracellular Matrix Deposition by Ascorbic Acid-Stimulated Human Corneal Fibroblasts

    PubMed Central

    Guo, Xiaoqing; Hutcheon, Audrey E. K.; Melotti, Suzanna A.; Zieske, James D.; Trinkaus-Randall, Vickery; Ruberti, Jeffrey W.

    2016-01-01

    Purpose To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures. Methods Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of Vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to five weeks. Constructs were assessed via light (phase contrast and differential interference contrast) and transmission (standard and quick freeze/deep etch) microscopy. Results Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27–51 nm) which often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct while quick-freeze deep etch micrographs showed the details of the matrix interaction with fibroblasts via arrays of membrane surface structures. Conclusions Human keratocytes, cultured in a stable Vitamin C derivative, are capable of assembling extracellular matrix which comprise parallel arrays of ECM fibrils. The resulting constructs, which are highly cellular, exhibit morphology similar to the developing mammalian stroma where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggest a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes which govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue-engineering a biomimetic stroma. PMID:17724187

  11. Sternal reconstruction by extracellular matrix: a rare case of phaces syndrome

    PubMed Central

    Molinaro, Francesco; Cerchia, Elisa; Di Crescenzo, Vincenzo Giuseppe; Luzzi, Luca; Bulotta, Anna Lavinia; Gotti, Giuseppe; Messina, Mario

    2016-01-01

    Abstract Congenital defects of the sternum are rare and due to a failure of midline development and fusion of the sternal bones. Surgical correction of a sternal cleft should be preferred during infancy for functional reasons. Chest wall reconstruction represented a complex problem in the last decades. We report our successful outcome of sternal reconstruction in a rare case of PHACES syndrome, in which the patient was submitted to reconstruction of the sternum and complete closure of the thoracic defect by the employ of an extracellular matrix XCM Biologic tissue matrix. We promote the use of extracellular matrix in surgical reconstruction of chest defects for its maneuverability, plasticity, tolerability and the possibility of growing with the children’s chest getting a good compliance and optimal cosmetic results.

  12. Impact of changes in extracellular matrix in the lumbar degenerative disc

    PubMed Central

    Ciurea, AV; Mitrica, M; Mohan, A

    2011-01-01

    The complexity of the clinical, biochemical, hystochemical and immunologic aspects of the intervertebral disk, along with its molecular biology, justifies the object of our study on the extracellular matrix modifications in lumbar disk hernias and their impact on patient quality of life. Material and method: the research lot was composed of 50 patients, aged between 18 and 73, who have undergone lumbar disk hernia surgery. MMP–9 (metalloproteinase–9) and TIMP–1 (tissue inhibitor of matrix metalloprotease 1) have been dosed in order to study the modifications on extracellular disk matrix, and quality of life assessment was carried out both in pre–operatory and post–operatory periods. Conclusions: patients may prevent the appearance of degenerative processes of the intervertebral disk with care and responsibility by controlling their weight, avoiding intense physical activities and ceasing to smoke. PMID:22567050

  13. Components, structure, biogenesis and function of the Hydra extracellular matrix in regeneration, pattern formation and cell differentiation.

    PubMed

    Sarras, Michael P

    2012-01-01

    The body wall of Hydra is organized as an epithelial bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM), termed mesoglea by early biologists. Morphological studies have determined that Hydra ECM is composed of two basal lamina layers positioned at the base of each epithelial layer with an intervening interstitial matrix. Molecular and biochemical analyses of Hydra ECM have established that it contains components similar to those seen in more complicated vertebrate species. These components include such macromolecules as laminin, type IV collagen, and various fibrillar collagens. These components are synthesized in a complicated manner involving cross-talk between the epithelial bilayer. Any perturbation to ECM biogenesis leads to a blockage in Hydra morphogenesis. Blockage in ECM/cell interactions in the adult polyp also leads to problems in epithelial transdifferentiation processes. In terms of biophysical parameters, Hydra ECM is highly flexible; a property that facilitates continuous movements along the organism's longitudinal and radial axis. This is in contrast to the more rigid matrices often found in vertebrates. The flexible nature of Hydra ECM can in part now be explained by the unique structure of the organism's type IV collagen and fibrillar collagens. This review will focus on Hydra ECM in regard to: 1) its general structure, 2) its molecular composition, 3) the biophysical basis for the flexible nature of Hydra's ECM, 4) the relationship of the biogenesis of Hydra ECM to regeneration of body form, and 5) the functional role of Hydra ECM during pattern formation and cell differentiation.

  14. Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment.

    PubMed

    Barone, L M; Faris, B; Chipman, S D; Toselli, P; Oakes, B W; Franzblau, C

    1985-06-18

    The protein composition in the extracellular matrix of cultured neonatal rat aortic smooth muscle cells has been monitored over time in culture. The influence of ascorbate on insoluble elastin and collagen has been described. In the absence of ascorbate, the cells accumulate an insoluble elastin component which can account for as much as 50% of the total protein in the extracellular matrix. In the presence of ascorbate, the amount of insoluble collagen increases, while the insoluble elastin content is significantly less. When ascorbate conditions are varied at different times during the culture, the extracellular matrices are altered with respect to collagen and elastin ratios. The decrease in elastin accumulation in the presence of ascorbate may be explained by an overhydroxylation of tropoelastin. Approximately 1/3 of the prolyl residues in the soluble elastin fractions isolated from cultures grown in the presence of ascorbate are hydroxylated. Since the insoluble elastin accumulated in these cultures contain the unique lysine-derived cross-links in amounts comparable to aortic tissue, this culture system proves ideal for studying the influence of extracellular matrix elastin on cell growth and metabolism.

  15. Activin A suppresses osteoblast mineralization capacity by altering extracellular matrix (ECM) composition and impairing matrix vesicle (MV) production.

    PubMed

    Alves, Rodrigo D A M; Eijken, Marco; Bezstarosti, Karel; Demmers, Jeroen A A; van Leeuwen, Johannes P T M

    2013-10-01

    During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP(1)) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.

  16. Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II

    PubMed Central

    Ruppender, Nazanin S.; Guo, Ruijing; Dadwal, Ushashi C.; Cannonier, Shellese; Basu, Sandip; Guelcher, Scott A.; Sterling, Julie A.

    2015-01-01

    Cancer patients frequently develop skeletal metastases that significantly impact quality of life. Since bone metastases remain incurable, a clearer understanding of molecular mechanisms regulating skeletal metastases is required to develop new therapeutics that block establishment of tumors in bone. While many studies have suggested that the microenvironment contributes to bone metastases, the factors mediating tumors to progress from a quiescent to a bone-destructive state remain unclear. In this study, we hypothesized that the “soil” of the bone microenvironment, specifically the rigid mineralized extracellular matrix, stimulates the transition of the tumor cells to a bone-destructive phenotype. To test this hypothesis, we synthesized 2D polyurethane (PUR) films with elastic moduli ranging from the basement membrane (70 MPa) to cortical bone (3800 MPa) and measured expression of genes associated with mechanotransduction and bone metastases. We found that expression of Integrin β3 (Iβ3), as well as tumor-produced factors associated with bone destruction (Gli2 and parathyroid hormone related protein (PTHrP)), significantly increased with matrix rigidity, and that blocking Iβ3 reduced Gli2 and PTHrP expression. To identify the mechanism by which Iβ3 regulates Gli2 and PTHrP (both are also known to be regulated by TGF-β), we performed Förster resonance energy transfer (FRET) and immunoprecipitation, which indicated that Iβ3 co-localized with TGF-β Receptor Type II (TGF-β RII) on rigid but not compliant films. Finally, transplantation of tumor cells expressing Iβ3 shRNA into the tibiae of athymic nude mice significantly reduced PTHrP and Gli2 expression, as well as bone destruction, suggesting a crucial role for tumor-produced Iβ3 in disease progression. This study demonstrates that the rigid mineralized bone matrix can alter gene expression and bone destruction in an Iβ3/TGF-β-dependent manner, and suggests that Iβ3 inhibitors are a potential

  17. Differential expression of extracellular matrix components in the Fallopian tubes throughout the menstrual cycle

    PubMed Central

    2012-01-01

    Background One of the unique characteristics of the female genital tract is the extensive tissue remodeling observed throughout the menstrual cycle. Multiple components of the extracellular matrix take part in this tissue rebuilding; however, the individual components involved have not been identified. Methods In the present study, the expression of extracellular matrix proteins and selected matrix metalloproteinase (MMP) activities in Fallopian tubes (FT) throughout the menstrual cycle were examined by PCR array, immunocytochemistry, zymography and bioinformatics. Results Of the eighty-four genes analyzed, eighty-three were expressed in the FT during at least one stage of the menstrual cycle. We observed a significant increase (>/=2-fold) in ADAMTS1, ADAMTS13, COL7A1, MMP3, MMP9, PECAM1, and THBS3 in the periovulatory phase compared to the follicular phase. Meanwhile, we observed a significant decrease (>/= 2-fold) in COL7A1, ICAM1, ITGA8, MMP16, MMP9, CLEC3B, SELE and TIMP2 in the lutheal phase compared to the periovulatory phase. Immunocytochemistry showed that MMP-3 and MMP-9 were localized in the endosalpinx during all phases of the menstrual cycle. Gelatin zymograms detected non-cycle-dependent protease activity. Conclusions Several extracellular matrix components were regulated throughout the menstrual cycle in a cyclic pattern, suggesting a possible steroid regulation and a role in tissue remodeling and FT functions. PMID:22897899

  18. Effect of spaceflight on the extracellular matrix of skeletal muscle after a crush injury

    NASA Technical Reports Server (NTRS)

    Stauber, W. T.; Fritz, V. K.; Burkovskaia, T. E.; Il'ina-Kakueva, E. I.

    1992-01-01

    The organization and composition of the extracellular matrix were studied in the crush-injured gastrocnemius muscle of rats subjected to 0 G. After 14 days of flight on Cosmos 2044, the gastrocnemius muscle was removed and evaluated by histochemical and immunohistochemical techniques from the five injured flight rodents and various earth-based treatment groups. In general, the repair process was similar in all injured muscle samples with regard to the organization of the extracellular matrix and myofibers. Small and large myofibers were present within an expanded extracellular matrix, indicative of myogenesis and muscle regeneration. In the tail-suspended animals, a more complete repair was observed with nonenlarged area of nonmuscle cells or matrix material visible. In contrast, the muscle samples from the flight animals were less well organized and contained more macrophages and blood vessels in the repair region, indicative of a delayed repair process, but did not demonstrate any chronic inflammation. Myofiber repair did vary in muscles from the different groups, being slowest in the flight animals and most complete in the tail-suspended ones.

  19. Effect of spaceflight on the extracellular matrix of skeletal muscle after a crush injury

    NASA Technical Reports Server (NTRS)

    Stauber, W. T.; Fritz, V. K.; Burkovskaia, T. E.; Il'ina-Kakueva, E. I.

    1992-01-01

    The organization and composition of the extracellular matrix were studied in the crush-injured gastrocnemius muscle of rats subjected to 0 G. After 14 days of flight on Cosmos 2044, the gastrocnemius muscle was removed and evaluated by histochemical and immunohistochemical techniques from the five injured flight rodents and various earth-based treatment groups. In general, the repair process was similar in all injured muscle samples with regard to the organization of the extracellular matrix and myofibers. Small and large myofibers were present within an expanded extracellular matrix, indicative of myogenesis and muscle regeneration. In the tail-suspended animals, a more complete repair was observed with nonenlarged area of nonmuscle cells or matrix material visible. In contrast, the muscle samples from the flight animals were less well organized and contained more macrophages and blood vessels in the repair region, indicative of a delayed repair process, but did not demonstrate any chronic inflammation. Myofiber repair did vary in muscles from the different groups, being slowest in the flight animals and most complete in the tail-suspended ones.

  20. An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to C. albicans1

    PubMed Central

    Byrd, Angel S.; O’Brien, Xian M.; Johnson, Courtney M.; Lavigne, Liz M.; Reichner, Jonathan S.

    2013-01-01

    The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as Neutrophil Extracellular Traps (NETs). The receptor:ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Since the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with ECM, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern β-glucan by human neutrophils causes rapid (≤ 30 mins) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized β-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn + β-glucan is robust, accounting for 17.2 ± 3.4% of total DNA in the cell population. Release is dependent on β-glucan recognition by CR3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on β-glucan recognition by CR3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid anti-fungal response. PMID:23509360

  1. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

    PubMed

    Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

  2. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  3. Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix.

    PubMed

    Stephenson, Matthew K; Lenihan, Sean; Covarrubias, Roman; Huttinger, Ryan M; Gumina, Richard J; Sawyer, Douglas B; Galindo, Cristi L

    2016-06-14

    Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

  4. Design of fibrin matrix composition to enhance endothelial cell growth and extracellular matrix deposition for in vitro tissue engineering.

    PubMed

    Pankajakshan, Divya; Krishnan, Lissy K

    2009-01-01

    Tissue-engineered blood vessel substitutes should closely resemble native vessels in terms of structure, composition, mechanical properties, and function. Successful cardiovascular tissue engineering requires optimization of in vitro culture environment that would produce functional constructs. The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system to render elasticity to blood vessel wall, whereas collagen is responsible for providing mechanical strength. The objective of this study was to understand the significance of various ECM components on endothelial cell (EC) growth and tissue generation. We demonstrate that, even though fibrin is a good matrix for EC growth, fibronectin is the crucial component of the fibrin matrix that enhances EC adhesion, spreading, and proliferation. Vascular EC growth factor is known to influence in vitro growth of EC, but, so far, ECM deposition in in vitro culture has not been reported. In this study, it is shown that incorporation of a mixture of hypothalamus-derived angiogenic growth factors with fibrin matrix enhances synthesis and deposition of insoluble elastin and collagen in the matrix, within 10 days of in vitro culture. The results suggest that a carefully engineered fibrin composite matrix may support EC growth, survival, and remodeling of ECM in vitro and impart optimum properties to the construct for resisting the shear stress at the time of implantation.

  5. Preliminary Experience in the Use of an Extracellular Matrix (CorMatrix) as a Tube Graft: Word of Caution.

    PubMed

    Hibino, Narutoshi; McConnell, Patrick; Shinoka, Toshiharu; Malik, Mahim; Galantowicz, Mark

    2015-01-01

    A number of materials have been used for the repair of congenital heart disease. However, an ideal material is yet to be discovered. Decellularized extracellular matrix from porcine small intestinal submucosa (CorMatrix) has been developed and commercialized as a biological tissue substitute. This has been used for valvuloplasty, sepal defect repair, or angioplasty as a patch. In this study, we demonstrate our preliminary experience using CorMatrix as a tube graft. A retrospective review of 13 patients who underwent cardiac surgery using CorMatrix as an interposition graft was performed (10 patients for central pulmonary artery reconstruction in comprehensive stage II surgery for hypoplastic left-sided heart syndrome and 3 patients for aortic arch reconstruction in interrupted aortic arch). At a mean follow-up of 9.7 months, 8 of 10 patients who underwent central pulmonary artery reconstruction using CorMatrix tube showed progressive significant stenosis. One patient underwent replacement of the CorMatrix tube with a homograft because of severe stenosis after the placement of a stent. All 3 patients who had aortic arch reconstruction with the CorMatrix tube demonstrated no stenosis, no dilatation, and no aneurysm formation. Although angioplasty using CorMatrix as an interposition tube vascular graft demonstrated no adverse event in the aortic position in short term, a high rate of intimal hyperplasia formation with significant stenosis was found in the low-pressure small-diameter system. Longer follow-up is required to assess the growth potential of the arterial conduit. CorMatrix may not be the ideal material as conduit in the low-pressure small-diameter system to provide long-term durable outcomes.

  6. Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

    PubMed

    Xie, Jing; Hou, Yanhua; Fu, Na; Cai, Xiaoxiao; Li, Guo; Peng, Qiang; Lin, Yunfeng

    2015-10-01

    Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones.

  7. Moderate cyclic tensile strain alters the assembly of cartilage extracellular matrix proteins in vitro.

    PubMed

    Bleuel, Judith; Zaucke, Frank; Brüggemann, Gert-Peter; Heilig, Juliane; Wolter, Marie-Louise; Hamann, Nina; Firner, Sara; Niehoff, Anja

    2015-06-01

    Mechanical loading influences the structural and mechanical properties of articular cartilage. The cartilage matrix protein collagen II essentially determines the tensile properties of the tissue and is adapted in response to loading. The collagen II network is stabilized by the collagen II-binding cartilage oligomeric matrix protein (COMP), collagen IX, and matrilin-3. However, the effect of mechanical loading on these extracellular matrix proteins is not yet understood. Therefore, the aim of this study was to investigate if and how chondrocytes assemble the extracellular matrix proteins collagen II, COMP, collagen IX, and matrilin-3 in response to mechanical loading. Primary murine chondrocytes were applied to cyclic tensile strain (6%, 0.5 Hz, 30 min per day at three consecutive days). The localization of collagen II, COMP, collagen IX, and matrilin-3 in loaded and unloaded cells was determined by immunofluorescence staining. The messenger ribo nucleic acid (mRNA) expression levels and synthesis of the proteins were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and western blots. Immunofluorescence staining demonstrated that the pattern of collagen II distribution was altered by loading. In loaded chondrocytes, collagen II containing fibrils appeared thicker and strongly co-stained for COMP and collagen IX, whereas the collagen network from unloaded cells was more diffuse and showed minor costaining. Further, the applied load led to a higher amount of COMP in the matrix, determined by western blot analysis. Our results show that moderate cyclic tensile strain altered the assembly of the extracellular collagen network. However, changes in protein amount were only observed for COMP, but not for collagen II, collagen IX, or matrilin-3. The data suggest that the adaptation to mechanical loading is not always the result of changes in RNA and/or protein expression but might also be the result of changes in matrix assembly and structure.

  8. Chondroitinase injection improves keloid pathology by reorganizing the extracellular matrix with regenerated elastic fibers.

    PubMed

    Ishiko, Toshihiro; Naitoh, Motoko; Kubota, Hiroshi; Yamawaki, Satoko; Ikeda, Mika; Yoshikawa, Katsuhiro; Fujita, Hiroshi; Yamaguchi, Hiroaki; Kurahashi, Yasuhiro; Suzuki, Shigehiko

    2013-05-01

    Keloids are a proliferative fibrotic disease characterized by abnormal accumulation of extracellular matrix in the dermis. Keloid lesions lack skin plasticity due to deficiencies in elastic fiber formation in the extracellular matrix. The loss of elastic fiber is caused by excessive accumulation of chondroitin sulfate (CS), a sulfated glycosaminoglycan. However, there is no radical cure for keloids. Using a model system, we show herein that treatment of keloid tissues with chondroitinase ABC, an enzyme that specifically digests CS, improves clinical features of keloids. Keloid tissues obtained from patients were grafted on nude mice, and chondroitinase ABC was injected into the grafted keloid tissues. Chondroitinase ABC treatment significantly reduced the volume of keloid implants concomitant with recovery of elastic fiber formation. These results suggest that chondroitinase ABC injection is an effective therapy for keloid. © 2013 Japanese Dermatological Association.

  9. How Osteoblasts Sense their Environment: Integrin-Extracellular Matrix Interactions and Mechanical Loading of Bone

    NASA Technical Reports Server (NTRS)

    Globus, Ruth K.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Osteoblasts are the cells responsible for forming and replacing bone throughout life. We know that mechanical stimulation through weight-bearing at I gravity on Earth is needed to maintain healthy bone, and that osteoblasts play a critical role in that process. Over the last 9 years in my laboratory at NASA ARC, we have studied the regulation of osteoblast function by interactions between the extracellular matrix and die cell. Using a cell culture approach, we defined the repertoire of adhesion receptors, called integrins, which are expressed on the osteoblast surface, as well as specific extracellular matrix proteins, which are needed for cellular differentiation and survival. We are now extending these observations to determine if integrin signaling is involved in the skeletal responses to disuse and recovery from disuse using the rodent model of hindlimb unloading by tail suspension. Together, our cell culture and animal studies are providing new insight into the regulation of osteoblast function in bone.

  10. Extracellular matrix modifications at fertilization: regulation of dityrosine crosslinking by transamidation

    PubMed Central

    Wong, Julian L.; Wessel, Gary M.

    2009-01-01

    Summary Fertilization is accompanied by the construction of an extracellular matrix that protects the new zygote. In sea urchins, this structure is built from glycoproteins residing at the egg surface and in secretory vesicles at the egg cortex. Four enzymatic activities are required for the transformation of these proteins into the mechanically and chemically resilient fertilization envelope: proteolysis, transamidation, NADPH-dependent oxidation and peroxidation. Here, we identify the Strongylocentrotus purpuratus enzymes responsible for the formation of ε(γ-glutamyl)lysine crosslinks (transamidation). We find that these two transglutaminases are activated by local acidification and act on specific substrates within the fertilization envelope (including ovoperoxidase, rendezvin and SFE9). Surprisingly, these enzymes also regulate dityrosine crosslinking both by direct conjugation of ovoperoxidase and by modulating hydrogen peroxide production. Together, these results emphasize how transglutaminases can coordinate the activities of other enzymes during extracellular matrix transmogrifications. PMID:19403662

  11. How Osteoblasts Sense their Environment: Integrin-Extracellular Matrix Interactions and Mechanical Loading of Bone

    NASA Technical Reports Server (NTRS)

    Globus, Ruth K.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Osteoblasts are the cells responsible for forming and replacing bone throughout life. We know that mechanical stimulation through weight-bearing at I gravity on Earth is needed to maintain healthy bone, and that osteoblasts play a critical role in that process. Over the last 9 years in my laboratory at NASA ARC, we have studied the regulation of osteoblast function by interactions between the extracellular matrix and die cell. Using a cell culture approach, we defined the repertoire of adhesion receptors, called integrins, which are expressed on the osteoblast surface, as well as specific extracellular matrix proteins, which are needed for cellular differentiation and survival. We are now extending these observations to determine if integrin signaling is involved in the skeletal responses to disuse and recovery from disuse using the rodent model of hindlimb unloading by tail suspension. Together, our cell culture and animal studies are providing new insight into the regulation of osteoblast function in bone.

  12. Role of oligomerization domains in thrombospondins and other extracellular matrix proteins.

    PubMed

    Engel, Jürgen

    2004-06-01

    Coiled coils, collagen triple helices and globular oligomerization domains mediate the subunit assembly of many proteins in vertebrates and invertebrates. Oligomerization offers functional advantages including multivalency, increased binding strength and the combined function of different domains. These features are seen in natural proteins and may be introduced by protein engineering. The special focus of this review is on oligomerization domain of extracellular matrix proteins. For thrombospondins, initial interesting results on the functional role of oligomerization have been published. Other features remain to be explored. For example, it is not clear why thrombospondin-1 and thrombospondin-2 are trimers whereas thrombospondins-3 to -5 are pentamers. To stimulate this type of research, this review makes a survey of oligomerization domains and their functional role in extracellular matrix proteins.

  13. Extracellular Matrix in Plants and Animals: Hooks and Locks for Viruses.

    PubMed

    Stavolone, Livia; Lionetti, Vincenzo

    2017-01-01

    The extracellular matrix (ECM) of animal and plants cells plays important roles in viral diseases. While in animal cells extracellular matrix components can be exploited by viruses for recognition, attachment and entry, the plant cell wall acts as a physical barrier to viral entry and adds a higher level of difficulty to intercellular movement of viruses. Interestingly, both in plant and animal systems, ECM can be strongly remodeled during virus infection, and the understanding of remodeling mechanisms and molecular players offers new perspectives for therapeutic intervention. This review focuses on the different roles played by the ECM in plant and animal hosts during virus infection with special emphasis on the similarities and differences. Possible biotechnological applications aimed at improving viral resistance are discussed.

  14. Extracellular Matrix in Plants and Animals: Hooks and Locks for Viruses

    PubMed Central

    Stavolone, Livia; Lionetti, Vincenzo

    2017-01-01

    The extracellular matrix (ECM) of animal and plants cells plays important roles in viral diseases. While in animal cells extracellular matrix components can be exploited by viruses for recognition, attachment and entry, the plant cell wall acts as a physical barrier to viral entry and adds a higher level of difficulty to intercellular movement of viruses. Interestingly, both in plant and animal systems, ECM can be strongly remodeled during virus infection, and the understanding of remodeling mechanisms and molecular players offers new perspectives for therapeutic intervention. This review focuses on the different roles played by the ECM in plant and animal hosts during virus infection with special emphasis on the similarities and differences. Possible biotechnological applications aimed at improving viral resistance are discussed. PMID:28955324

  15. Extracellular Matrix Remodeling During the Progression of Volume Overload-Induced Heart Failure

    PubMed Central

    Hutchinson, Kirk R.; Stewart, James A.; Lucchesi, Pamela A.

    2009-01-01

    Volume overload-induced heart failure results in progressive left ventricular remodeling characterized by chamber dilation, eccentric cardiac myocyte hypertrophy and changes in extracellular matrix (ECM) remodeling changes. The ECM matrix scaffold is an important determinant of the structural integrity of the myocardium and actively participates in force transmission across the LV wall. In response to this hemodynamic overload, the ECM undergoes a distinct pattern of remodeling that differs from pressure overload. Once thought to be a static entity, the ECM is now regarded to be a highly adaptive structure that is dynamically regulated by mechanical stress, neurohormonal activation, inflammation and oxidative stress, that result in alterations in collagen and other matrix components and a net change in matrix metalloproteinase (MMP) expression and activation. These changes dictate overall ECM turnover during volume overload hear failure progression. This review will discuss the cellular and molecular mechanisms that dictate the temporal patterns of ECM remodeling during heart disease progression. PMID:19524591

  16. MT1-MMP regulates the turnover and endocytosis of extracellular matrix fibronectin.

    PubMed

    Shi, Feng; Sottile, Jane

    2011-12-01

    The extracellular matrix (ECM) is dynamically remodeled by cells during development, normal tissue homeostasis and in a variety of disease processes. We previously showed that fibronectin is an important regulator of ECM remodeling. The deposition and/or polymerization of fibronectin into the ECM controls the deposition and stability of other ECM molecules. In addition, agents that inhibit fibronectin polymerization promote the turnover of fibronectin fibrils and enhance ECM fibronectin endocytosis and intracellular degradation. Endocytosis of ECM fibronectin is regulated by β1 integrins, including α5β1 integrin. We have examined the role of extracellular proteases in regulating ECM fibronectin turnover. Our data show that membrane type matrix metalloproteinase 1 (MT1-MMP; also known as MMP14) is a crucial regulator of fibronectin turnover. Cells lacking MT1-MMP show reduced turnover and endocytosis of ECM fibronectin. MT1-MMP regulates ECM fibronectin remodeling by promoting extracellular cleavage of fibronectin and by regulating α5β1-integrin endocytosis. Our data also show that fibronectin polymerization stabilizes fibronectin fibrils and inhibits ECM fibronectin endocytosis by inhibiting α5β1-integrin endocytosis. These data are the first to show that an ECM protein and its modifying enzyme can regulate integrin endocytosis. These data also show that integrin trafficking plays a major role in modulating ECM fibronectin remodeling. The dual dependence of ECM fibronectin turnover on extracellular proteolysis and endocytosis highlights the complex regulatory mechanisms that control ECM remodeling to ensure maintenance of proper tissue function.

  17. The nephrotoxic Ifosfamide-metabolite chloroacetaldehyde interferes with renal extracellular matrix homeostasis.

    PubMed

    Benesic, Andreas; Schwerdt, Gerald; Hennemeier, Isabell; Sauvant, Christoph; Mildenberger, Sigrid; Gekle, Michael

    2014-01-01

    Chronic renal proximal tubule dysfunction after therapy with the antineoplastic agent ifosfamide (IFO) is often attributed to the metabolite chloroacetaldehyde (CAA). Chronic IFO-nephropathy is reported to result in tubulointerstitial fibrosis and inflammation. To elucidate possible effects of CAA on extracellular matrix homeostasis, we investigated the action of CAA on markers of extracellular matrix (ECM) homeostasis in human proximal tubule cells (RPTEC) by use of direct ELISA for extracellular collagens and gelatin zymography. An increase in type III collagen and a decrease in type IV collagen abundance in the media of RPTEC could be observed after exposure to CAA in clinically relevant concentrations. CAA increased intracellular type III and decreased intracellular type IV collagen. MMP-2 activity was decreased but MMP-9 activity unchanged. The enhanced CAA-induced collagen III formation could be attenuated by the intracellular Ca(2+)-chelator BAPTA-AM, the PKA-antagonist H-89 and by extracellular acidification. CAA-induced collagen III abundance was enhanced by db-cAMP and IBMX and by protein overload. CAA exerts profibrotic effects on RPTEC dependent on Ca(2+) and cAMP/PKA-signaling. These effects are enhanced by additional protein burden and attenuated by acidification. © 2014 S. Karger AG, Basel. © 2014 S. Karger AG, Basel.

  18. Utilization of a tubularized cormatrix extracellular matrix for repair of an arteriovenous fistula aneurysm.

    PubMed

    DuBose, Joseph J; Azizzadeh, Ali

    2015-02-01

    Salvage of failing autogenous arteriovenous fistula (AVF) access sites is a primary concern in dialysis access preservation and a key emphasis of the Kidney Disease Outcomes Quality Initiative. Aneurysmal degeneration of autogenenous fistulae represents a significant complication, associated with potential skin breakdown, rupture, infection, and access loss. The utilization of conduits derived from extracellular matrix (ECM), currently approved for cardiovascular repairs, may mitigate some of the risks associated with salvage using synthetic materials. We report the utilization of a tubularized CorMatrix(®) ECM graft as a novel conduit for repair of a brachiocephalic AVF aneurysm. Published by Elsevier Inc.

  19. Contribution of the α8 integrin chain to the expression of extracellular matrix components.

    PubMed

    Volkert, Gudrun; Jahn, Angelika; Dinkel, Christina; Fahlbusch, Fabian; Zürn, Christina; Hilgers, Karl F; Rascher, Wolfgang; Hartner, Andrea; Marek, Ines

    2014-04-01

    In the kidney, the α8 integrin chain (itga8) is expressed in mesenchymal cells and is upregulated in fibrotic disease. We hypothesized that itga8 mediates a profibrotic phenotype of renal cells by promoting extracellular matrix and cytokine expression. Genetic itga8 deficiency caused complex changes in matrix expression patterns in mesangial and smooth-muscle cells, with the only concordant effect in both cell types being a reduction of collagen III expression. Silencing of itga8 with siRNA led to a decline of matrix turnover with repression of matrix metalloproteinases and reduction of matrix production. In contrast, de novo expression of itga8 in tubular epithelial cells resulted in reduced collagen synthesis. Overexpression of itga8 in fibroblasts did not change the expression of matrix molecules or regulators of matrix turnover. Thus, the influence of itga8 on the expression of matrix components was not uniform and celltype dependent. Itga8 seems unlikely to exert overall profibrotic effects in renal cells.

  20. Cell-extracellular matrix interactions in the ductus arteriosus and perinatal pulmonary circulation.

    PubMed

    Rabinovitch, M

    1996-12-01

    Our studies and those of others have shown that changes in the extracellular matrix have profound effects on vascular remodeling. In the ductus, increased production of endothelial hyaluronan and smooth muscle cell chondroitin sulfate and fibronectin and impaired elastin fiber assembly are features critical to smooth muscle cell migration into the subendothelium and intimal cushion formation. There is a developmentally orchestrated process that involves post-transcriptional mechanisms of gene regulation. Closure of the ductus arteriosus is associated with further changes in matrix expression and programmed cell death. The changes in the extracellular matrix that induce neointimal formation are also observed in pathological conditions in pulmonary and coronary arteries. Plasticity of the pulmonary circulation in the perinatal period also involves matrix regulation, and processes that prevent the normal decrease in pulmonary vascular resistance will result in impaired matrix regulation, and in the development of structural changes in the pulmonary arteries, including abnormal smooth muscle cell differentiation, hypertrophy and proliferation, which sustain the elevation in pulmonary artery pressure.

  1. Differential expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in avian tibial dyschondroplasia.

    PubMed

    Shahzad, Muhammad; Liu, Jingying; Gao, Jianfeng; Wang, Zhi; Zhang, Ding; Nabi, Fazul; Li, Kun; Li, Jiakui

    2015-01-01

    Tibial dyschondroplasia (TD) is an avian bone disorder of different aetiologies that may be associated with lameness. The disorder is characterized by focal disruption of endochondral bone formation, with a lack of matrix proteolysis and an accumulation of non-mineralized avascular cartilage. The aim of this study was to determine the expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in normal, thiram-induced TD lesions and in the process of recovery from TD in broiler chickens. An extracellular matrix (ECM) degrading enzyme, matrix metalloproteinase-9 (MMP-9), was selected to investigate the effects of CD147 in the degradation of ECM. Gene expression was analysed by quantitative real-time polymerase chain reaction and protein levels by immunohistochemistry and western blotting. The birds were divided into three groups: thiram fed; recovery; and controls. Genes encoding CD147 and MMP-9 were down-regulated during the development of the disease, and were up-regulated during recovery. Western blotting also showed lower protein levels of CD147 in TD, which increased during the recovery phase associated with ECM degradation and growth plate repair. The findings of this study suggest that ECM has a crucial role in the occurrence of TD and that CD147 appears to play a pivotal role in matrix proteolysis in the chicken, similar to that in other species.

  2. The extracellular matrix in breast cancer predicts prognosis through composition, splicing, and crosslinking.

    PubMed

    Robertson, Claire

    2016-04-10

    The extracellular matrix in the healthy breast has an important tumor suppressive role, whereas the abnormal ECM in tumors can promote aggressiveness, and has been linked to breast cancer relapse, survival and resistance to chemotherapy. This review article gives an overview of the elements of the ECM which have been linked to prognosis of breast cancers, including changes in ECM protein composition, splicing, and microstructure.

  3. Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

    NASA Technical Reports Server (NTRS)

    Ehrhart, E. J.; Gillette, E. L.; Barcellos-Hoff, M. H.; Chaterjee, A. (Principal Investigator)

    1996-01-01

    High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

  4. [Preparation and characterization of an extracellular matrix of artificial tendon tissue from natural macromolecules].

    PubMed

    Xiong, Yanfei; Wan, Li

    2008-11-01

    Collagen and chitosan are well natrual polymers to be used as extracellular matrix on tissue engineering because of their biocompatibility, certain mechanical strength and biodegradability. But there are some disadvantages when they are used to construct extracellular matrix respectively. This experiment utilized their complementary performances to prepare a composite extracellular matrix of artificial tendon tissue that had adequacy mechanics strength and good biocompatibility, cell affinity, biodegradability. Collagen and chitosan were covalently crosslinked using EDC and NHS to obtain a porous scaffold material that the porous was oriented under an external force. Then RGD peptide was covalently attached to scaffold material surface to improve its affinity with cells. The microstructure of scaffold material was observed under microscope and scanning electron microscope. Simultaneously, the physical performance, hydrophilicity, ecto-degradation rate and cell compatibility of scaffold material were measured in the experiments. The results showed that this scaffold material was soft and stretchy. Its tensile strength was 15.0 MPa, corresponding shape extension was 7.33%, and its porosity was 79.4%. Its water absorption rate and water retention rate were 772% and 206% respectively. Its degradation rate in RPM 1640 culture mediun with 10% fetal bovine serum and in human serum were 4.13% and 37.2% respectively after three weeks. These degradation rates are suitable for the rehab course of injured tendon. Moreover the degradation rates can be controlled by adjusting technological conditions and degree of cross linking. Significantly higher affinity with 3T3-Ll cell was detected on the scaffold material modified by RGD peptide. The various physical performances of this complex scaffold material are appropriate for constructing extracellular matrix of artificial tendon tissue or artificial skin. Moreover, it could be used as soft tissue slurry for plastic surgery.

  5. The extracellular matrix of muscle--implications for manipulation of the craniofacial musculature.

    PubMed

    Lewis, M P; Machell, J R; Hunt, N P; Sinanan, A C; Tippett, H L

    2001-08-01

    Successful adaptation of craniofacial skeletal muscle is dependent upon the connective tissue component of the muscle. This is exemplified by procedures such as distraction histo/osteogenesis. The mechanisms underlying remodelling of intramuscular connective tissue are complex and multifactorial and involve extracellular matrix (ECM) molecules, receptors for the ECM (integrins) and enzymes that remodel the ECM (MMPs). This review discusses the current state of knowledge and clinical implications of connective tissue biology as applied to craniofacial skeletal muscle.

  6. Study Of The Efficacy Of Extracellular Matrix Arterial Interposition Grafts In A Sheep (Ovis aries) Model

    DTIC Science & Technology

    2015-07-29

    interposition grafts in a sheep (Ovis aries ) model." PRINCIPAL INVESTIGATOR (Pl) I TRAINING COORDINATOR (TC): Maj Lucas Neff DEPARTMENT: General...USAGE: Animal Species: Total# Approved # Used this FY Total# Used to Date Ovies aries 12 8 4 2. PROTOCOL TYPE I CHARACTERISTICS: (Check all applicable...and conclusions/applications.) Title: Study of the efficacy of extracellular matrix arterial interposition grafts in a sheep (Ovis aries ) model

  7. Repair of a penetrating ascending aortic ulcer with localized resection and extracellular matrix patch aortoplasty.

    PubMed

    Smith, Craig R; Stamou, Sotiris C; Boeve, Theodore J; Hooker, Robert C

    2012-09-01

    Penetrating ascending aortic ulcers are rarely encountered, yet they present significant risk of hemorrhage and aortic dissection. Expedient recognition and repair is of vital importance. The current management of penetrating ulcer of the ascending aorta includes replacement of the ascending aorta with a prosthetic graft. We describe our technique of repairing a penetrating ulcer of the ascending aorta with localized ulcer resection and extracellular matrix patch aortoplasty.

  8. Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

    SciTech Connect

    Ehrhart, E.J.; Gillette, E.L.; Barcellos-Hoff, M.H.

    1996-02-01

    High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by {gamma} irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin inummoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation. 40 refs., 3 figs.

  9. Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

    NASA Technical Reports Server (NTRS)

    Ehrhart, E. J.; Gillette, E. L.; Barcellos-Hoff, M. H.; Chaterjee, A. (Principal Investigator)

    1996-01-01

    High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

  10. Escherichia coli biofilms have an organized and complex extracellular matrix structure.

    PubMed

    Hung, Chia; Zhou, Yizhou; Pinkner, Jerome S; Dodson, Karen W; Crowley, Jan R; Heuser, John; Chapman, Matthew R; Hadjifrangiskou, Maria; Henderson, Jeffrey P; Hultgren, Scott J

    2013-09-10

    Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community. Bacteria can form biofilms in diverse niches, including abiotic surfaces, living cells, and at the air-liquid interface of liquid media. Encasing these cellular communities is a self-produced extracellular matrix (ECM) that can be composed of proteins, polysaccharides, and nucleic acids. The ECM protects biofilm bacteria from environmental insults and also makes the dissolution of biofilms very challenging. As a result, formation of biofilms within humans (during infection) or on industrial material (such as water pipes) has detrimental and costly effects. In order to combat bacterial biofilms, a better understanding of components required for biofilm formation and the ECM is required. This study defined the ECM composition and architecture of floating pellicle biofilms formed by Escherichia coli.

  11. Thrombospondin-1 contributes to slower aortic aneurysm growth by inhibiting maladaptive remodeling of extracellular matrix.

    PubMed

    Satoh, Mamoru; Nasu, Takahito; Osaki, Takuya; Hitomi, Sho

    2017-06-01

    In this issue of Clinical Science, Krishna and colleagues describe recent work on thrombospondin-1 (TSP-1) maturation and its association with slower growth of aortic aneurysm in TSP-1 knockdown mouse models. The authors conclude that TSP-1 deficiency promotes maladaptive remodeling of the extracellular matrix (ECM) leading to accelerated aortic aneurysm progression. We comment on a causal relation between TSP-1 and the progression of aortic aneurysm. © 2017 The Author(s).

  12. The Role Of Cells, Neurotrophins, Extracellular Matrix And Cell Surface Molecules In Peripheral Nerve Regeneration

    PubMed Central

    Naidu, Murali

    2009-01-01

    Wallerian degeneration is a complicated process whereby axons and myelin sheaths undergo degeneration, and eventually are phagocytosed by macrophages and Schwann cells following nerve damage. Schwann cells proliferate and the endoneural tubes persist. In addition, neurotrophins, neural cell adhesion molecules, cytokines and other soluble factors are upregulated to facilitate regeneration. The important role of cellular components, neurotrophins, and extracellular matrix components, including cell surface molecules involved in this regenerative process, is highlighted and discussed in this review. PMID:22589652

  13. Tumors perturbing extracellular matrix biosynthesis. The case of von Recklinghausen's disease.

    PubMed

    Robert, L

    2014-04-01

    This is a short review of neurofibromatosis-1 or von Recklinghausen's disease, due to a loss of function mutation of the gene neurofibromin-1, which normally inhibits the Ras MAPK-pathways. Among its symptoms, the strong oversynthesis of several collagen types designates this disease as producing a deregulation of extracellular matrix biosynthesis involved in tumor formation. Up to about 40% of the skin tumors consist of collagens. A short summary of the clinical manifestations and pathological and genetic mechanisms are also described.

  14. Extracellular matrix remodeling in wound healing of critical size defects in the mitral valve leaflet.

    PubMed

    Stephens, Elizabeth H; Nguyen, Tom C; Blazejewski, Jack G; Vekilov, Dragoslava P; Connell, Jennifer P; Itoh, Akinobu; Ingels, Neil B; Miller, D Craig; Grande-Allen, K Jane

    2016-07-01

    The details of valvular leaflet healing following valvuloplasty and leaflet perforation from endocarditis are poorly understood. In this study, the synthesis and turnover of valvular extracellular matrix due to healing of a critical sized wound was investigated. Twenty-nine sheep were randomized to either CTRL (n = 11) or HOLE (n = 18), in which a 2.8-4.8 mm diameter hole was punched in the posterior mitral leaflet. After 12 weeks, posterior leaflets were harvested and histologically stained to localize extracellular matrix components. Immunohistochemistry was also performed to assess matrix components and markers of matrix turnover. A semi-quantitative grading scale was used to quantify differences between HOLE and CTRL. After 12 weeks, the hole diameter was reduced by 71.3 ± 1.4 % (p < 0.001). Areas of remodeling surrounding the hole contained more activated cells, greater expression of proteoglycans, and markers of matrix turnover (prolyl 4-hydroxylase, metalloproteases, and lysyl oxidase, each p ≤ 0.025), along with fibrin accumulation. Two distinct remodeling regions were evident surrounding the hole, one directly bordering the hole rich in versican and hyaluronan and a second adjacent region with abundant collagen and elastic fiber turnover. The remodeling also caused reduced delineation between valve layers (p = 0.002), more diffuse staining of matrix components and markers of matrix turnover (p < 0.001), and disruption of the collagenous fibrosa. In conclusion, acute valve injury elicited distinct, heterogeneous alterations in valvular matrix composition and structure, resulting in partial wound closure. Because these changes could also affect leaflet mechanics and valve function, it will be important to determine their impact on healing wounds.

  15. Augmentation of Achilles tendon repair with extracellular matrix xenograft: a biomechanical analysis.

    PubMed

    Magnussen, Robert A; Glisson, Richard R; Moorman, Claude T

    2011-07-01

    Achilles tendon rupture is a frequent injury in athletes and the general public. Cases of chronic rupture or poor tendon quality secondary to tendinopathy are challenging to repair primarily. Commercially available extracellular matrix materials have been utilized in recent years to augment tendon repair. Augmentation of Achilles tendon with extracellular matrix xenograft results in reduced repair site gapping and increased peak failure load in a cadaveric model featuring simulated physiologic loads. Controlled laboratory study. Ten matched pairs of fresh-frozen human lower extremities amputated just below the knee were obtained and each Achilles tendon was sharply tenotomized. One randomly selected specimen from each matched pair underwent Achilles repair using a 4-strand Krackow technique with extracellular matrix xenograft augmentation (TissueMend Soft Tissue Repair Matrix), while the opposite tendon underwent suture repair alone as a control. Each tendon was then subjected to 1000 sinusoidal tensile loading cycles to 86 N during which repair site gapping was monitored, followed by distraction to failure. One pair was used to evaluate the effects of graft orientation and not included in the analysis. Significantly less gapping was noted in the augmented tendon group at all time points after the 10th load cycle (P < .05). The mean repair site gapping after 1000 cycles of loading was 4.0 mm (range, 3.1-5.0 mm) in the augmented group and 6.5 mm (range, 4.1-8.6 mm) in the suture-only group. The ultimate failure load was 821 N (range, 613-1021 N) in the augmented group and 392 N (range, 322-481 N) in the suture-only group (P < .01). The augmentation of Achilles tendon repair with extracellular matrix xenograft decreases gapping and increases load to failure immediately after surgery in a cadaveric model. Tendon repair augmentation may allow more aggressive early rehabilitation, particularly in cases of chronic rupture or poor tendon quality. Further work is necessary

  16. Highly Conserved Salt Bridge Stabilizes Rigid Signal Patch at Extracellular Loop Critical for Surface Expression of Acid-sensing Ion Channels*

    PubMed Central

    Yang, Yang; Yu, Ye; Cheng, Jin; Liu, Yan; Liu, Di-Shi; Wang, Jin; Zhu, Michael X.; Wang, Rui; Xu, Tian-Le

    2012-01-01

    Acid-sensing ion channels (ASICs) are non-selective cation channels activated by extracellular acidosis associated with many physiological and pathological conditions. A detailed understanding of the mechanisms that govern cell surface expression of ASICs, therefore, is critical for better understanding of the cell signaling under acidosis conditions. In this study, we examined the role of a highly conserved salt bridge residing at the extracellular loop of rat ASIC3 (Asp107-Arg153) and human ASIC1a (Asp107-Arg160) channels. Comprehensive mutagenesis and electrophysiological recordings revealed that the salt bridge is essential for functional expression of ASICs in a pH sensing-independent manner. Surface biotinylation and immunolabeling of an extracellular epitope indicated that mutations, including even minor alterations, at the salt bridge impaired cell surface expression of ASICs. Molecular dynamics simulations, normal mode analysis, and further mutagenesis studies suggested a high stability and structural constrain of the salt bridge, which serves to separate an adjacent structurally rigid signal patch, important for surface expression, from a flexible gating domain. Thus, we provide the first evidence of structural requirement that involves a stabilizing salt bridge and an exposed rigid signal patch at the destined extracellular loop for normal surface expression of ASICs. These findings will allow evaluation of new strategies aimed at preventing excessive excitability and neuronal injury associated with tissue acidosis and ASIC activation. PMID:22399291

  17. Cell-Derived Polymer/Extracellular Matrix Composite Scaffolds for Cartilage Regeneration, Part 1: Investigation of Cocultures and Seeding Densities for Improved Extracellular Matrix Deposition

    PubMed Central

    Levorson, Erica J.; Mountziaris, Paschalia M.; Hu, Olivia; Kasper, F. Kurtis

    2014-01-01

    This study investigated the coculture of chondrocytes and mesenchymal stem cells (MSCs) on electrospun fibrous polymer scaffolds to produce polymer/extracellular matrix (ECM) hybrid constructs with the objective of reducing the number of chondrocytes necessary to produce ample cartilage-like ECM within the scaffolds. To generate these hybrid constructs, electrospun poly(ɛ-caprolactone) fibrous scaffolds were seeded at both high and low initial densities with five different ratios of chondrocytes to MSCs: 1:0, 1:1, 1:3, 1:5, and 0:1, and cultured for 7, 14, and 21 days. Glycosaminoglycan production and distribution within the three coculture groups was similar to quantities generated by chondrocyte-only controls. Conversely, as the concentration of chondrocytes was increased, the collagen content of the constructs also increased at each time point, with a 1:1 chondrocyte to MSC ratio approximating the collagen production of chondrocytes alone. Histological staining suggested that cocultured constructs mimicked the well-distributed ECM patterns of chondrocyte generated constructs, while improving greatly over the restricted distribution of matrix within MSC-only constructs. These results support the capacity of cocultures of chondrocytes and MSCs to generate cartilaginous matrix within a polymeric scaffold. Further, the inclusion of MSCs in these cocultures enables the reduction of chondrocytes needed to produce cell-generated ECM. PMID:24007559

  18. Relationships between melanocytes, mechanical properties and extracellular matrix composition in mouse heart valves.

    PubMed

    Carneiro, Flavia; Kruithof, Boudewijn Pt; Balani, Kanthesh; Agarwal, Arvind; Gaussin, Vinciane; Kos, Lidia

    2015-01-01

    Heart valves are complex structures composed of organized layers of extracellular matrix, and interstitial and overlying endothelial cells. In this article, we present the specific localization of a population of melanocytes within the murine heart valves at ages important for their post-natal development. In all stages analyzed in our study, melanocytes were found in high numbers populating the atrial aspect of the tricuspid and mitral leaflets. The pulmonary valve did not present melanocytes. To characterize a putative role for the valve melanocytes, the dynamic nanomechanical properties of tricuspid leaftets containing large numbers or no melanocytes were measured. The stiffness coefficient of hyperpigmented leaflets was higher (11.5 GPa) than the ones from wild-type (7.5 GPa) and hypopigmented (5.5 GPa) leaflets. These results suggest that melanocytes may contribute to the mechanical properties of the heart valves. The arrangement of extracellular matrix molecules such as Collagen I and Versican B is responsible for the mechanical characteristics of the leaflets. Melanocytes were found to reside primarily in areas of Versican B expression. The patterns of expression of Collagen I and Versican B were not, however, disrupted in hyper or hypopigmented leaflets. Melanocytes may affect other extracellular matrix molecules to alter the valves' microenvironment.

  19. Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix

    PubMed Central

    Seminara, Agnese; Wilking, James N.; Vlamakis, Hera; Ebrahim, Senan; Kolter, Roberto; Weitz, David A.; Brenner, Michael P.

    2012-01-01

    Bacterial biofilms are organized communities of cells living in association with surfaces. The hallmark of biofilm formation is the secretion of a polymeric matrix rich in sugars and proteins in the extracellular space. In Bacillus subtilis, secretion of the exopolysaccharide (EPS) component of the extracellular matrix is genetically coupled to the inhibition of flagella-mediated motility. The onset of this switch results in slow expansion of the biofilm on a substrate. Different strains have radically different capabilities in surface colonization: Flagella-null strains spread at the same rate as wild type, while both are dramatically faster than EPS mutants. Multiple functions have been attributed to the EPS, but none of these provides a physical mechanism for generating spreading. We propose that the secretion of EPS drives surface motility by generating osmotic pressure gradients in the extracellular space. A simple mathematical model based on the physics of polymer solutions shows quantitative agreement with experimental measurements of biofilm growth, thickening, and spreading. We discuss the implications of this osmotically driven type of surface motility for nutrient uptake that may elucidate the reduced fitness of the matrix-deficient mutant strains. PMID:22232655

  20. Targeting the extracellular matrix of ovarian cancer using functionalized, drug loaded lyophilisomes.

    PubMed

    van der Steen, Sophieke C H A; Raavé, René; Langerak, Sjoerd; van Houdt, Laurens; van Duijnhoven, Sander M J; van Lith, Sanne A M; Massuger, Leon F A G; Daamen, Willeke F; Leenders, William P; van Kuppevelt, Toin H

    2017-04-01

    Epithelial ovarian cancer is characterized by a high mortality rate and is in need for novel therapeutic avenues to improve patient outcome. The tumor's extracellular matrix ("stroma") offers new possibilities for targeted drug-delivery. Recently we identified highly sulfated chondroitin sulfate (CS-E) as a component abundantly present in the ovarian cancer extracellular matrix, and as a novel target for anti-cancer therapy. Here, we report on the functionalization of drug-loaded lyophilisomes (albumin-based biocapsules) to specifically target the stroma of ovarian carcinomas with the potential to eliminate cancer cells. To achieve specific targeting, we conjugated single chain antibodies reactive with CS-E to lyophilisomes using a two-step approach comprising sortase-mediated ligation and bioorthogonal click chemistry. Antibody-functionalized lyophilisomes specifically targeted the ovarian cancer stroma through CS-E. In a CS-E rich micro-environment in vitro lyophilisomes induced cell death by extracellular release of doxorubicin which localized to the nucleus. Immunohistochemistry identified CS-E rich stroma in a variety of solid tumors other than ovarian cancer, including breast, lung and colon cancer indicating the potential versatility of matrix therapy and the use of highly sulfated chondroitin sulfates in cancer stroma as a micro-environmental hook for targeted drug delivery. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Coming into focus: the role of extracellular matrix in vertebrate optic cup morphogenesis.

    PubMed

    Kwan, Kristen M

    2014-10-01

    The vertebrate eye acquires its basic form during the process of optic cup morphogenesis, during which the optic vesicle emerges from the brain neuroepithelium and, through a series of cell and tissue movements, transforms itself into the multilayered optic cup, containing neural retina (comprised of retinal progenitors), retinal pigmented epithelium, and the lens, which is derived from the overlying ectoderm. While great strides have been made to understand the developmental signals controlling specification, patterning, and differentiation of the optic cup, only in recent years have the cellular and molecular bases of optic cup morphogenesis begun to be unraveled. One critical component of the morphogenetic process is the extracellular matrix: the complex, glycoprotein-rich layer that surrounds the optic vesicle and lens. Though the extracellular matrix has long been visualized by classical histological techniques and postulated to play various roles in optic cup development, its functional role was uncertain. This is now beginning to change, as live imaging techniques, quantitative image analyses, molecular genetics and in vitro models yield new insights into the process of optic cup morphogenesis and the specific influences of particular extracellular matrix components and their associated signaling pathways.

  2. Exposure of cardiac fibroblasts to the herbicide nitrofen causes altered interactions with the extracellular matrix.

    PubMed

    Borck, A; Massey, E; Loftis, M J; Carver, W

    2004-02-01

    A large proportion of congenital heart defects result from dysmorphogenesis of valvuloseptal precursors, the endocardial cushions. Intrinsic to formation and maturation of these tissues are developmental changes in cell-cell and cell-extracellular matrix interactions. Interactions between cells and the extracellular matrix play critical roles in modulating cellular processes including proliferation, migration, differentiation and even survival. While significant progress is being made in the elucidation of the cellular events involved in valvuloseptal development, little is known regarding how environmental factors may affect this process. Embryonic exposure to the herbicide nitrofen has been shown to result in congenital heart defects associated with altered endocardial cushion formation or maturation. The present studies were performed to begin to address the cellular mechanisms of these nitrofen-induced effects. Heart fibroblasts were isolated and treated with varying doses of nitrofen in vitro. Experiments were performed to determine the effects of this herbicide on important cellular processes including migration, proliferation and apoptosis. These studies illustrated a dose-dependent decrease in collagen gel contraction and proliferation in response to nitrofen. Assays were also performed to determine the effects of nitrofen on fibroblast gene expression. Increased expression of collagen type I and specific integrins were seen following nitrofen exposure. These studies illustrate that nitrofen has direct effects on cardiac fibroblast proliferation and extracellular matrix remodeling, cellular events important in valvuloseptal development.

  3. Quantitation and relative distribution of extracellular matrix in Staphylococcus epidermidis biofilm

    SciTech Connect

    Van Pett, K.; Schurman, D.J.; Smith, R.L. )

    1990-05-01

    The relationship between adherence of bacteria to foreign bodies and their deposition of extracellular matrix was examined on glass and suture material. To quantitate bacterial adherence, uptake of ({sup 3}H)thymidine into bacterial DNA was analyzed. Corresponding amounts of extracellular matrix were measured by a new technique using ({sup 14}C)glucose incorporation. This study shows that ({sup 14}C)glucose preferentially labeled bacterial strains in proportion to biofilm production. The ratio of {sup 3}H{sup 14}C in high biofilm producers was 0.9 and in low producers it was 3.7. Radioactive identification of organisms as high and low producers was confirmed by electron microscopy. The results presented here show that production and accumulation of biofilm over time is a stable characteristic in different strains of S. epidermidis. The use of ratios reflecting radiolabeling of bacteria and biofilm by ({sup 3}H)thymidine and ({sup 14}C)glucose, respectively, is a quantitative yet simple technique to assess extracellular matrix of different strains of S. epidermidis.

  4. Interaction between the extracellular matrix and lymphatics - consequences for lymphangiogenesis and lymphatic function

    PubMed Central

    Wiig, Helge; Keskin, Doruk; Kalluri, Raghu

    2014-01-01

    The lymphatic system is important for body fluid balance as well as immunological surveillance. Due to the identification of new molecular markers during the last decade, there has been a recent dramatic increase in our knowledge on the molecular mechanisms involved in lymphatic vessel growth (lymphangiogenesis) and lymphatic function. Here we review data showing that although it is often overlooked, the extracellular matrix plays an important role in the generation of new lymphatic vessels as a response to physiological and pathological stimuli. Extracellular matrix-lymphatic interactions as well as biophysical characteristics of the stroma have consequences for tumor formation, growth and metastasis. During the recent years, anti-lymphangiogenesis has emerged as an additional therapeutic modality to the clinically applied anti-angiogenesis strategy. Oppositely, enhancement of lymphangiogenesis in situations of lymph accumulation is seen as a promising strategy to a set of conditions where few therapeutic avenues are available. Knowledge on the interaction between the extracellular matrix and the lymphatics may enhance our understanding of the underlying mechanisms and may ultimately lead to better therapies for conditions where reduced or increased lymphatic function is the therapeutic target PMID:20727409

  5. Aggrecan-based extracellular matrix is an integral part of the human basal ganglia circuit.

    PubMed

    Brückner, G; Morawski, M; Arendt, T

    2008-01-24

    The extracellular matrix is known to be involved in neuronal communication and the regulation of plastic changes, and also considered to protect neurons and synapses against damage. The goal of this study was to investigate how major extracellular matrix components (aggrecan, link protein, hyaluronan) constitute the pathways of the nigral system in the human basal ganglia circuit affected by neurodegeneration in Parkinson's disease. Here we show that aggrecan- and link protein-related components form clear regional distribution patterns, whereas hyaluronan is widely distributed in gray and white matter. Two predominant phenotypes of the aggrecan-based matrix can be discriminated: (1) perineuronal nets (PNs) and (2) axonal coats (ACs) encapsulating preterminal fibers and synaptic boutons. Clearly contoured PNs are associated with GABAergic projection neurons in the external and internal division of the globus pallidus, the lateral and reticular part of the substantia nigra, as well as subpopulations of striatal and thalamic inhibitory interneurons. Dopaminergic nigral neurons are devoid of PNs but are contacted to a different extent by matrix-coated boutons forming subnucleus-specific patterns. A very dense network of ACs is characteristic especially of the posterior lateral cell groups of the compact substantia nigra (nigrosome 1). In the subthalamic nucleus and the lateral thalamic nuclei numerous AC-associated axons were attached to principal neurons devoid of PNs. We conclude from the region-specific patterns that the aggrecan-based extracellular matrix is adapted to the fast processing of sensorimotor activities which are the therapeutic target of surgery and deep brain stimulation in the treatment of advanced stages of Parkinson's disease.

  6. Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix

    PubMed Central

    Bishop, Christopher A.; Best, Michael; Rich, Celeste B.; Stone, Phillip J.

    2017-01-01

    Smooth muscle cells contribute to extracellular matrix remodeling during atherogenesis. De-differentiated, synthetic smooth muscle cells are involved in processes of migration, proliferation and changes in expression of extracellular matrix components, all of which contribute to loss of homeostasis accompanying atherogenesis. Elevated levels of acute phase proteins, including serum amyloid A (SAA), are associated with an increased risk for atherosclerosis. Although infection with periodontal and respiratory pathogens via activation of inflammatory cell Toll-like receptor (TLR)2 has been linked to vascular disease, little is known about smooth muscle cell TLR2 in atherosclerosis. This study addresses the role of SAA and TLR2 activation on smooth muscle cell matrix gene expression and insoluble elastin accumulation. Cultured rat aortic smooth muscle cells were treated with SAA or TLR2 agonists and the effect on expression of matrix metallopeptidase 9 (MMP9) and tropoelastin studied. SAA up-regulated MMP9 expression. Tropoelastin is an MMP9 substrate and decreased tropoelastin levels in SAA-treated cells supported the concept of extracellular matrix remodeling. Interestingly, SAA-induced down-regulation of tropoelastin was not only evident at the protein level but at the level of gene transcription as well. Contributions of proteasomes, nuclear factor κ B and CCAAT/enhancer binding protein β on regulation of MMP9 vs. tropoleastin expression were revealed. Effects on Mmp9 and Eln mRNA expression persisted with long-term SAA treatment, resulting in decreased insoluble elastin accumulation. Interestingly, the SAA effects were TLR2-dependent and TLR2 activation by bacterial ligands also induced MMP9 expression and decreased tropoelastin expression. These data reveal a novel mechanism whereby SAA and/or infection induce changes in vascular elastin consistent with atherosclerosis. PMID:28257481

  7. Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs.

    PubMed

    Jabłońska-Trypuć, Agata; Matejczyk, Marzena; Rosochacki, Stanisław

    2016-01-01

    The main group of enzymes responsible for the collagen and other protein degradation in extracellular matrix (ECM) are matrix metalloproteinases (MMPs). Collagen is the main structural component of connective tissue and its degradation is a very important process in the development, morphogenesis, tissue remodeling, and repair. Typical structure of MMPs consists of several distinct domains. MMP family can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs. MMPs and their inhibitors have multiple biological functions in all stages of cancer development: from initiation to outgrowth of clinically relevant metastases and likewise in apoptosis and angiogenesis. MMPs and their inhibitors are extensively examined as potential anticancer drugs. MMP inhibitors can be divided into two main groups: synthetic and natural inhibitors. Selected synthetic inhibitors are in clinical trials on humans, e.g. synthetic peptides, non-peptidic molecules, chemically modified tetracyclines, and bisphosphonates. Natural MMP inhibitors are mainly isoflavonoids and shark cartilage.

  8. Cartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: interaction, regulation and role in chondrogenesis.

    PubMed

    Acharya, Chitrangada; Yik, Jasper H N; Kishore, Ashleen; Van Dinh, Victoria; Di Cesare, Paul E; Haudenschild, Dominik R

    2014-07-01

    Thrombospondins (TSPs) are widely known as a family of five calcium-binding matricellular proteins. While these proteins belong to the same family, they are encoded by different genes, regulate different cellular functions and are localized to specific regions of the body. TSP-5 or Cartilage Oligomeric Matrix Protein (COMP) is the only TSP that has been associated with skeletal disorders in humans, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). The pentameric structure of COMP, the evidence that it interacts with multiple cellular proteins, and the recent reports of COMP acting as a 'lattice' to present growth factors to cells, inspired this review of COMP and its interacting partners. In our review, we have compiled the interactions of COMP with other proteins in the cartilage extracellular matrix and summarized their importance in maintaining the structural integrity of cartilage as well as in regulating cellular functions.

  9. Functional Ultrasound Imaging for Assessment of Extracellular Matrix Scaffolds Used for Liver Organoid Formation

    PubMed Central

    Gessner, Ryan C.; Hanson, Ariel D.; Feingold, Steven; Cashion, Avery T.; Corcimaru, Ana; Wu, Bryant T.; Mullins, Christopher R.; Aylward, Stephen R.; Reid, Lola M.; Dayton, Paul A.

    2015-01-01

    A method of 3D functional ultrasound imaging has been developed to enable non-destructive assessment of extracellular matrix scaffolds that have been prepared by decellularization protocols and are intended for recellularization to create organoids. A major challenge in organ decellularization is retaining patent micro-vascular structures crucial for nutrient access and functionality of organoids. The imaging method described here provides statistical distributions of flow rates throughout the tissue volumes, 3D vessel network architecture visualization, characterization of microvessel volumes and sizes, and delineation of matrix from vascular circuits. The imaging protocol was tested on matrix scaffolds that are tissue-specific, but not species-specific, matrix extracts, prepared by a process that preserved >98% of the collagens, collagen-associated matrix components, and matrix-bound growth factors and cytokines. Image-derived data are discussed with respect to assessment of scaffolds followed by proof-of-concept studies in organoid establishment using Hep3B, human hepatoblast-like cells. Histology showed that the cells attached to scaffolds with patent vasculature within minutes, achieved engraftment at near 100%, expressed liver-specific functions within 24h, and yielded evidence of proliferation and increasing differentiation of cells throughout the two weeks of culture studies. This imaging method should prove valuable in analyses of such matrix scaffolds. PMID:24011714

  10. Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy.

    PubMed

    Reichhardt, Courtney; Ferreira, Jose A G; Joubert, Lydia-Marie; Clemons, Karl V; Stevens, David A; Cegelski, Lynette

    2015-11-01

    Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional (15)N and (31)P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.

  11. Induction of Extracellular Matrix-Remodeling Genes by the Senescence-Associated Protein APA-1

    PubMed Central

    Benanti, Jennifer A.; Williams, Dawnnica K.; Robinson, Kristin L.; Ozer, Harvey L.; Galloway, Denise A.

    2002-01-01

    Human fibroblasts undergo cellular senescence after a finite number of divisions, in response to the erosion of telomeres. In addition to being terminally arrested in the cell cycle, senescent fibroblasts express genes that are normally induced upon wounding, including genes that remodel the extracellular matrix. We have identified the novel zinc finger protein APA-1, whose expression increased in senescent human fibroblasts independent of telomere shortening. Extended passage, telomerase-immortalized fibroblasts had increased levels of APA-1 as well as the cyclin-dependent kinase inhibitor p16. In fibroblasts, APA-1 was modified by the ubiquitin-like protein SUMO-1, which increased APA-1 half-life, possibly by blocking ubiquitin-mediated degradation. Overexpression of APA-1 did not cause cell cycle arrest; but, it induced transcription of the extracellular matrix-remodeling genes MMP1 and PAI2, which are associated with fibroblast senescence. MMP1 and PAI2 transcript levels also increased in telomerase-immortalized fibroblasts that had high levels of APA-1, demonstrating that the matrix-remodeling phenotype of senescent fibroblasts was not induced by telomere attrition alone. APA-1 was able to transactivate and bind to the MMP1 promoter, suggesting that APA-1 is a transcription factor that regulates expression of matrix-remodeling genes during fibroblast senescence. PMID:12370286

  12. Subepithelial fibrosis and degradation of the bronchial extracellular matrix in cystic fibrosis.

    PubMed

    Durieu, I; Peyrol, S; Gindre, D; Bellon, G; Durand, D V; Pacheco, Y

    1998-08-01

    Cystic fibrosis is a genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator gene. Chronic inflammation and proteolysis lead to progressive damage of the bronchial wall. Extracellular matrix determines the structural organization and the mechanical properties of lung airways. It was thus examined in nine patients with cystic fibrosis (six bronchial biopsies and three lobectomies) in order to assess its level of alteration. The submucosal changes in matrix protein distribution were analyzed by immunochemistry and electron microscopy: the subepithelial basal lamina was thinned; an acellular collagen fiber layer composed of interstitial collagens (types I and III) subtended by tenascin and devoid of elastin-associated microfibrils was deposited beneath the basal lamina; this dense fibrous deposit generally formed a thick layer and could extend into the bronchial wall; the bronchial elastic framework lost arborescent distribution and appeared slender, packed, or lacunar; ultrastructural observation gave evidence for elastic and collagenic fiber lysis. Proteolytic activity is probably the major cause of matrix degradation. Fibrosis appears as a repair process rather than as an active fibrogenesis. The reversibility of extracellular matrix alterations is an important challenge and various interventions such as anti-inflammatory treatments can be targeted to halt or reverse this degradation process.

  13. Extracellular matrix interacts with interferon {alpha} protein: Retention and display of cytotoxicity

    SciTech Connect

    Yoshida, Kimiko; Kondoh, Atsushi; Narumi, Kenta; Yoshida, Teruhiko; Aoki, Kazunori

    2008-11-14

    We have been investigating the efficacy of an intratumoral interferon (IFN)-{alpha} gene transfer against solid cancers, and found that when the gene is transduced into the subcutaneous tumors, IFN-{alpha} concentration is markedly increased in the injected tumor but not in the serum. To explain this effective confinement of IFN-{alpha} to target tissues, we hypothesized that the extracellular matrix in the tumors interacts with IFN-{alpha}. In this study, a solid-phase-binding assay and immunoprecipitation demonstrated that the IFN-{alpha} binds directly to matrix proteins. Immunohistochemical staining showed a co-localization of IFN-{alpha} with pericellular fibronectin. In addition, matrix-bound IFN-{alpha} protein transduced intracellular signaling and potentiated its cytotoxic activity, suggesting that the retention of IFN-{alpha} protein on extracellular matrix is likely to play a role in its in vivo biological activity. The data suggest a therapeutic advantage of the intratumoral IFN-{alpha} gene transfer over the conventional parenteral therapy both in the safety and efficacy.

  14. Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes.

    PubMed

    Visser, Michelle B; Pollitt, Christopher C

    2010-03-15

    Most research to date involving laminins and extracellular matrix protein function in both normal and pathological conditions involves in vitro culture of keratinocytes. Few methods are established to allow for prolonged propagation of keratinocytes from equine tissues, including the hoof lamellae. In this study we modified cell isolation and culture techniques to allow for proliferation and sub-culturing of equine lamellar keratinocytes. Additionally, the production and processing of extracellular matrix molecules by skin and lamellar keratinocytes were studied. Physical and proteolytic tissue separation in combination with media containing a calcium concentration of 0.6 mM in combination with additional media supplements proved optimal for proliferation and subculture of equine lamellar keratinocytes on collagen coated substratum. Immunofluorescence and immunoblotting studies confirmed that equine skin and lamellar keratinocytes produce Ln-332 in vitro and processing of this molecule follows that of other species. As well, matrix components including integrin alpha-6 (alpha 6) and the hemidesmosome proteins, bullous pemphigoid antigen 1 (BP180) bullous pemphigoid antigen 2 (BP230) and plectin are also expressed. Isolation of equine keratinocytes and study of the matrix and adhesion related molecules produced by them provides a valuable tool for future work in the veterinary field.

  15. Electrospinning adipose tissue-derived extracellular matrix for adipose stem cell culture.

    PubMed

    Francis, Michael P; Sachs, Patrick C; Madurantakam, Parthasarathy A; Sell, Scott A; Elmore, Lynne W; Bowlin, Gary L; Holt, Shawn E

    2012-07-01

    Basement membrane-rich extracellular matrices, particularly murine sarcoma-derived Matrigel, play important roles in regenerative medicine research, exhibiting marked cellular responses in vitro and in vivo, although with limited clinical applications. We find that a human-derived matrix from lipoaspirate fat, a tissue rich in basement membrane components, can be fabricated by electrospinning and used to support cell culture. We describe practical applications and purification of extracellular matrix (ECM) from adipose tissue (At-ECM) and its use in electrospinning scaffolds and adipose stem cell (ASC) culture. The matrix composition of this purified and electrospun At-ECM was assessed histochemically for basement membrane, connective tissue, collagen, elastic fibers/elastin, glycoprotein, and proteoglycans. Each histochemical stain was positive in fat tissue, purified At-ECM, and electrospun At-ECM, and to some extent positive in a 10:90 blend with polydioxanone (PDO). We also show that electrospun At-ECM, alone and blended with PDO, supports ASC attachment and growth, suggesting that electrospun At-ECM scaffolds support ASC cultivation. These studies show that At-ECM can be isolated and electrospun as a basement membrane-rich tissue engineering matrix capable of supporting stem cells, providing the groundwork for an array of future regenerative medicine advances.

  16. Extracellular Matrix Fibronectin Stimulates the Self-Assembly of Microtissues on Native Collagen Gels

    PubMed Central

    Sevilla, Carlos A.; Dalecki, Diane

    2010-01-01

    Fibronectin is an adhesive glycoprotein that is polymerized into extracellular matrices via a tightly regulated, cell-dependent process. Here, we demonstrate that fibronectin matrix polymerization induces the self-assembly of multicellular structures in vitro, termed tissue bodies. Fibronectin-null mouse embryonic fibroblasts adherent to compliant gels of polymerized type I collagen failed to spread or proliferate. In contrast, addition of fibronectin to collagen-adherent fibronectin-null mouse embryonic fibroblasts resulted in a dose-dependent increase in cell number, and induced the formation of three-dimensional (3D) multicellular structures that remained adherent and well-spread on the native collagen substrate. An extensive fibrillar fibronectin matrix formed throughout the microtissue. Blocking fibronectin matrix polymerization inhibited both cell proliferation and microtissue formation, demonstrating the importance of fibronectin fibrillogenesis in triggering cellular self-organization. Cell proliferation, tissue body formation, and tissue body shape were dependent on both fibronectin and collagen concentrations, suggesting that the relative proportion of collagen and fibronectin fibrils polymerized into the extracellular matrix influences the extent of cell proliferation and the final shape of microtissues. These data demonstrate a novel role for cell-mediated fibronectin fibrillogenesis in the formation and vertical assembly of microtissues, and provide a novel approach for engineering complex tissue architecture. PMID:20673131

  17. Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

    PubMed Central

    Reichhardt, Courtney; Ferreira, Jose A. G.; Joubert, Lydia-Marie; Clemons, Karl V.; Stevens, David A.

    2015-01-01

    Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The 13C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional 15N and 31P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions. PMID:26163318

  18. Development of an extracellular matrix delivery system for effective intramyocardial injection in ischemic tissue.

    PubMed

    Slaughter, Mark S; Soucy, Kevin G; Matheny, Robert G; Lewis, Beecher C; Hennick, Michael F; Choi, Young; Monreal, Gretel; Sobieski, Michael A; Giridharan, Guruprasad A; Koenig, Steven C

    2014-01-01

    Biomaterials with direct intramyocardial injection devices have been developed and are being investigated as a potential cardiac regenerative therapy for end-stage ischemic heart failure. Decellularized extracellular matrix (ECM) has been shown to improve cardiac function and attenuate or reverse pathologic remodeling cascades. CorMatrix Cardiovascular, Inc. has developed a porcine small intestinal submucosa-derived particulate extracellular matrix (P-ECM) and ECM Delivery System to provide uniform and controlled intramyocardial delivery of the injectable P-ECM material into infarcted regions. The CorMatrix ECM Delivery System is composed of a Multi-Needle P-ECM Syringe Assembly, Automated Injection Controller, and Tissue Depth Measurement System (portable ultrasound). Feasibility of the P-ECM delivery system was tested intraoperatively in a chronic ischemic heart failure bovine model (n = 11), and demonstrated the ability to control injection volume (0.1-1.0 ml) and depth of penetration (3-5 mm) under regulated injection pressure (150 psi CO2) into the ischemic region. Targeted intramyocardial delivery of P-ECM may improve efficacy and enable development of novel patient-specific therapy.

  19. Phenotypic diversity of neoplastic chondrocytes and extracellular matrix gene expression in cartilaginous neoplasms.

    PubMed Central

    Aigner, T.; Dertinger, S.; Vornehm, S. I.; Dudhia, J.; von der Mark, K.; Kirchner, T.

    1997-01-01

    Chondrocyte differentiation is characterized by distinct cellular phenotypes, which can be identified by specific extracellular matrix gene expression profiles. By applying in situ analysis on the mRNA and protein level in a series of benign and malignant human chondrogenic neoplasms, we were able to identify for the first time different phenotypes of neoplastic chondrocytes in vivo: 1) mature chondrocytes, which synthesized the characteristic cartilaginous extracellular tumor matrix, 2) cells resembling hypertrophic chondrocytes of the fetal growth plate, 3) cells resembling so-called dedifferentiated chondrocytes, and 4) well differentiated chondrocytic cells, which expressed type I collagen, indicating the presence of post-hypertrophic differentiated neoplastic chondrocytes. Chondrocytes exhibiting a range of phenotypes were found to be present in the same neoplasm. The different observed phenotypes, including the dedifferentiated phenotype, were in contrast to the anaplastic cells of high-grade chondrosarcomas. Comparison of expression data with tumor morphology revealed a relationship between the cellular phenotypes, the tumor matrix composition, and the matrix and cell morphology within the neoplasms. The distinctly different phenotypes of neoplastic chondrocytes are the basis of the characteristic high biochemical and morphological heterogeneity of chondroid neoplasms and shed light on their biological and clinical behavior. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9176404

  20. Engineering strategies to recapitulate epithelial morphogenesis within synthetic three-dimensional extracellular matrix with tunable mechanical properties

    NASA Astrophysics Data System (ADS)

    Miroshnikova, Y. A.; Jorgens, D. M.; Spirio, L.; Auer, M.; Sarang-Sieminski, A. L.; Weaver, V. M.

    2011-04-01

    The mechanical properties (e.g. stiffness) of the extracellular matrix (ECM) influence cell fate and tissue morphogenesis and contribute to disease progression. Nevertheless, our understanding of the mechanisms by which ECM rigidity modulates cell behavior and fate remains rudimentary. To address this issue, a number of two and three-dimensional (3D) hydrogel systems have been used to explore the effects of the mechanical properties of the ECM on cell behavior. Unfortunately, many of these systems have limited application because fiber architecture, adhesiveness and/or pore size often change in parallel when gel elasticity is varied. Here we describe the use of ECM-adsorbed, synthetic, self-assembling peptide (SAP) gels that are able to recapitulate normal epithelial acini morphogenesis and gene expression in a 3D context. By exploiting the range of viscoelasticity attainable with these SAP gels, and their ability to recreate native-like ECM fibril topology with minimal variability in ligand density and pore size, we were able to reconstitute normal and tumor-like phenotypes and gene expression patterns in nonmalignant mammary epithelial cells. Accordingly, this SAP hydrogel system presents the first tunable system capable of independently assessing the interplay between ECM stiffness and multi-cellular epithelial phenotype in a 3D context. Originally submitted for the special focus issue on physical oncology.

  1. Forces and torques on rigid inclusions in an elastic environment: Resulting matrix-mediated interactions, displacements, and rotations

    NASA Astrophysics Data System (ADS)

    Puljiz, Mate; Menzel, Andreas M.

    2017-05-01

    Embedding rigid inclusions into elastic matrix materials is a procedure of high practical relevance, for instance, for the fabrication of elastic composite materials. We theoretically analyze the following situation. Rigid spherical inclusions are enclosed by a homogeneous elastic medium under stick boundary conditions. Forces and torques are directly imposed from outside onto the inclusions or are externally induced between them. The inclusions respond to these forces and torques by translations and rotations against the surrounding elastic matrix. This leads to elastic matrix deformations, and in turn results in mutual long-ranged matrix-mediated interactions between the inclusions. Adapting a well-known approach from low-Reynolds-number hydrodynamics, we explicitly calculate the displacements and rotations of the inclusions from the externally imposed or induced forces and torques. Analytical expressions are presented as a function of the inclusion configuration in terms of displaceability and rotateability matrices. The role of the elastic environment is implicitly included in these relations. That is, the resulting expressions allow a calculation of the induced displacements and rotations directly from the inclusion configuration, without having to explicitly determine the deformations of the elastic environment. In contrast to the hydrodynamic case, compressibility of the surrounding medium is readily taken into account. We present the complete derivation based on the underlying equations of linear elasticity theory. In the future, the method will, for example, be helpful to characterize the behavior of externally tunable elastic composite materials, to accelerate numerical approaches, as well as to improve the quantitative interpretation of microrheological results.

  2. Forces and torques on rigid inclusions in an elastic environment: Resulting matrix-mediated interactions, displacements, and rotations.

    PubMed

    Puljiz, Mate; Menzel, Andreas M

    2017-05-01

    Embedding rigid inclusions into elastic matrix materials is a procedure of high practical relevance, for instance, for the fabrication of elastic composite materials. We theoretically analyze the following situation. Rigid spherical inclusions are enclosed by a homogeneous elastic medium under stick boundary conditions. Forces and torques are directly imposed from outside onto the inclusions or are externally induced between them. The inclusions respond to these forces and torques by translations and rotations against the surrounding elastic matrix. This leads to elastic matrix deformations, and in turn results in mutual long-ranged matrix-mediated interactions between the inclusions. Adapting a well-known approach from low-Reynolds-number hydrodynamics, we explicitly calculate the displacements and rotations of the inclusions from the externally imposed or induced forces and torques. Analytical expressions are presented as a function of the inclusion configuration in terms of displaceability and rotateability matrices. The role of the elastic environment is implicitly included in these relations. That is, the resulting expressions allow a calculation of the induced displacements and rotations directly from the inclusion configuration, without having to explicitly determine the deformations of the elastic environment. In contrast to the hydrodynamic case, compressibility of the surrounding medium is readily taken into account. We present the complete derivation based on the underlying equations of linear elasticity theory. In the future, the method will, for example, be helpful to characterize the behavior of externally tunable elastic composite materials, to accelerate numerical approaches, as well as to improve the quantitative interpretation of microrheological results.

  3. Decellularization of porcine skeletal muscle extracellular matrix for the formulation of a matrix hydrogel: a preliminary study.

    PubMed

    Fu, Yuehe; Fan, Xuejiao; Tian, Chunxiang; Luo, Jingcong; Zhang, Yi; Deng, Li; Qin, Tingwu; Lv, Qing

    2016-04-01

    Extracellular matrix (ECM) hydrogels are used as scaffolds to facilitate the repair and reconstruction of tissues. This study aimed to optimize the decellularization process of porcine skeletal muscle ECM and to formulate a matrix hydrogel scaffold. Five multi-step methods (methods A-E) were used to generate acellular ECM from porcine skeletal muscle [rinsing in SDS, trypsin, ethylenediaminetetraacetic acid (EDTA), Triton X-100 and/or sodium deoxycholate at 4-37°C]. The resulting ECM was evaluated using haematoxylin and eosin, 4-6-diamidino-2-phenylindole (DAPI) staining, and DNA quantification. Acellular matrix was dissolved in pepsin and gelled at 37°C. Hydrogel response to temperature was observed in vivo and in vitro. ECM components were assessed by Masson, Sirius red, and alcian blue staining, and total protein content. Acellular porcine skeletal muscle exhibited a uniform translucent white appearance. No intact nuclear residue was detected by haematoxylin and eosin staining, while DAPI staining showed a few nuclei in the matrixes produced by methods B, C, and D. Method A generated a gel that was too thin for gelation. However, the matrix obtained by rinsing in 0.2% trypsin/0.1% EDTA, 0.5% Triton X-100, and 1% Triton X-100/0.2% sodium deoxycholate was nuclei-free and produced a viscous solution that formed a structurally stable white jelly-like hydrogel. The residual DNA content of this solution was 49.37 ± 0.72 ng/mg, significantly less than in fresh skeletal muscle, and decreased to 19.22 ± 0.85 ng/mg after gelation (P < 0.05). The acellular matrix was rich in collagen and glycosaminoglycan, with a total protein concentration of 64.8 ± 6.9%. An acellular ECM hydrogel from porcine skeletal muscle was efficiently produced.

  4. Age-related macular degeneration and changes in the extracellular matrix

    PubMed Central

    Nita, Małgorzata; Strzałka-Mrozik, Barbara; Grzybowski, Andrzej; Mazurek, Urszula; Romaniuk, Wanda

    2014-01-01

    Age-related macular degeneration (AMD) is the leading cause of permanent, irreversible, central blindness (scotoma in the central visual field that makes reading and writing impossible, stereoscopic vision, recognition of colors and details) in patients over the age of 50 years in European and North America countries, and an important role is attributed to disorders in the regulation of the extracellular matrix (ECM). The main aim of this article is to present the crucial processes that occur on the level of Bruch’s membrane, with special consideration of the metalloproteinase substrates, metalloproteinase, and tissue inhibitor of metalloproteinase (TIMP). A comprehensive review of the literature was performed through MEDLINE and PubMed searches, covering the years 2005–2012, using the following keywords: AMD, extracellular matrix, metalloproteinases, tissue inhibitors of metalloproteinases, Bruch’s membrane, collagen, elastin. In the pathogenesis of AMD, a significant role is played by collagen type I and type IV; elastin; fibulin-3, -5, and -6; matrix metalloproteinase (MMP)-2, MMP-9, MMP-14, and MMP-1; and TIMP-3. Other important mechanisms include: ARMS2 and HTR1 proteins, the complement system, the urokinase plasminogen activator system, and pro-renin receptor activation. Continuous rebuilding of the extracellular matrix occurs in both early and advanced AMD, simultaneously with the dysfunction of retinal pigment epithelium (RPE) cells and endothelial cells. The pathological degradation or accumulation of ECM structural components are caused by impairment or hyperactivity of specific MMPs/TIMPs complexes, and is also endangered by the influence of other mechanisms connected with both genetic and environmental factors. PMID:24938626

  5. Mechanical phenotyping of cells and extracellular matrix as grade and stage markers of lung tumor tissues.

    PubMed

    Panzetta, Valeria; Musella, Ida; Rapa, Ida; Volante, Marco; Netti, Paolo A; Fusco, Sabato

    2017-07-15

    The mechanical cross-talk between cells and the extra-cellular matrix (ECM) regulates the properties, functions and healthiness of the tissues. When this is disturbed it changes the mechanical state of the tissue components, singularly or together, and cancer, along with other diseases, may start and progress. However, the bi-univocal mechanical interplay between cells and the ECM is still not properly understood. In this study we show how a microrheology technique gives us the opportunity to evaluate the mechanics of cells and the ECM at the same time. The mechanical phenotyping was performed on the surgically removed tissues of 10 patients affected by adenocarcinoma of the lung. A correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Our findings suggest a sort of asymmetric modification of the mechanical properties of the cells and the extra-cellular matrix in the tumor, being the more compliant cell even though it resides in a stiffer matrix. Overall, the simultaneous mechanical characterization of the tissues constituents (cells and ECM) provided new support for diagnosis and offered alternative points of analysis for cancer mechanobiology. When the integrity of the mechanical cross-talk between cells and the extra-cellular matrix is disturbed cancer, along with other diseases, may initiate and progress. Here, we show how a new technique gives the opportunity to evaluate the mechanics of cells and the ECM at the same time. It was applied on surgically removed tissues of 10 patients affected by adenocarcinoma of the lung and a correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  6. The Extracellular Matrix of the Lateral Pharyngeal Wall in Obstructive Sleep Apnea

    PubMed Central

    Dantas, Danielle Andrade da Silva; Mauad, Thais; Silva, Luiz F. F.; Lorenzi-Filho, Geraldo; Formigoni, Gilberto G. S.; Cahali, Michel B.

    2012-01-01

    Study Objectives: To compare the components of the extracellular matrix in the lateral pharyngeal muscular wall in patients with and without obstructive sleep apnea (OSA). This may help to explain the origin of the increased collapsibility of the pharynx in patients with OSA. Design: Specimens from the superior pharyngeal constrictor muscle, obtained during pharyngeal surgeries, were evaluated using histochemical and immunohistochemical analyses to determine the fractional area of collagen types I and III, elastic fibers, versican, fibronectin, and matrix metalloproteinases 1 and 2 in the endomysium. Setting: Academic tertiary center. Patiens: A total of 51 nonobese adult patients, divided into 38 patients with OSA and 13 nonsnoring control subjects without OSA. Interventions: Postintervention study performed on tissues from patients after elective surgery. Measurements and Results: Pharyngeal muscles of patients with OSA had significantly more collagen type I than pharyngeal muscles in control subjects. Collagen type I was correlated positively and independently with age. The other tested components of the extracellular matrix did not differ significantly between groups. In a logistic regression, an additive effect of both the increase of collagen type I and the increase in age with the presence of OSA was observed (odds ratio (OR), 2.06; 95% confidence interval (CI), 1.17-3.63), when compared with the effect of increased age alone (OR, 1.11; 95% CI, 1.03-1.20). Conclusion: Collagen type I in the superior pharyngeal constrictor muscle was more prevalent in patients with OSA and also increased with age. It was hypothesized that this increase could delay contractile-relaxant responses in the superior pharyngeal constrictor muscle at the expiratory-inspiratory phase transition, thus increasing pharyngeal collapsibility. Citation: Dantas DAS; Mauad T; Silva LFF; Lorenzi-Filho G; Formigoni GGS; Cahali MB. The extracellular matrix of the lateral pharyngeal wall in

  7. Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.

    PubMed

    Monet-Leprêtre, Marie; Haddad, Iman; Baron-Menguy, Céline; Fouillot-Panchal, Maï; Riani, Meriem; Domenga-Denier, Valérie; Dussaule, Claire; Cognat, Emmanuel; Vinh, Joelle; Joutel, Anne

    2013-06-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross

  8. Host-Parasite Interaction: Parasite-Derived and -Induced Proteases That Degrade Human Extracellular Matrix

    PubMed Central

    Piña-Vázquez, Carolina; Reyes-López, Magda; Ortíz-Estrada, Guillermo; de la Garza, Mireya; Serrano-Luna, Jesús

    2012-01-01

    Parasitic protozoa are among the most important pathogens worldwide. Diseases such as malaria, leishmaniasis, amoebiasis, giardiasis, trichomoniasis, and trypanosomiasis affect millions of people. Humans are constantly threatened by infections caused by these pathogens. Parasites engage a plethora of surface and secreted molecules to attach to and enter mammalian cells. The secretion of lytic enzymes by parasites into host organs mediates critical interactions because of the invasion and destruction of interstitial tissues, enabling parasite migration to other sites within the hosts. Extracellular matrix is a complex, cross-linked structure that holds cells together in an organized assembly and that forms the basement membrane lining (basal lamina). The extracellular matrix represents a major barrier to parasites. Therefore, the evolution of mechanisms for connective-tissue degradation may be of great importance for parasite survival. Recent advances have been achieved in our understanding of the biochemistry and molecular biology of proteases from parasitic protozoa. The focus of this paper is to discuss the role of protozoan parasitic proteases in the degradation of host ECM proteins and the participation of these molecules as virulence factors. We divide the paper into two sections, extracellular and intracellular protozoa. PMID:22792442

  9. Role of Extracellular Matrix Proteins and Their Receptors in the Development of the Vertebrate Neuromuscular Junction

    PubMed Central

    Singhal, Neha; Martin, Paul T.

    2012-01-01

    The vertebrate neuromuscular junction remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the neuromuscular junction, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins has been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic extracellular matrix proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability and transmission. PMID:21766463

  10. Extracellular matrix gene expression profiling using microfluidics for colorectal carcinoma stratification

    PubMed Central

    Hayes, Christopher J.; Dowling, Catriona M.; Dwane, Susan; McCumiskey, Mary E.; Tormey, Shona M.; Anne Merrigan, B.; Coffey, John C.; Kiely, Patrick A.; Dalton, Tara M.

    2016-01-01

    In cancer, biomarkers have many potential applications including generation of a differential diagnosis, prediction of response to treatment, and monitoring disease progression. Many molecular biomarkers have been put forward for different diseases but most of them do not possess the required specificity and sensitivity. A biomarker with a high sensitivity has a low specificity and vice versa. The inaccuracy of the biomarkers currently in use has led to a compelling need to identify more accurate markers with diagnostic and prognostic significance. The aim of the present study was to use a novel, droplet-based, microfluidic platform to evaluate the prognostic value of a panel of thirty-four genes that regulate the composition of extracellular matrices in colorectal carcinoma. Our method is a novel approach as it uses using continuous-flowing Polymerase Chain Reaction for the sensitive detection and accurate quantitation of gene expression. We identified a panel of relevant extracellular matrix genes whose expression levels were measured by real-time quantitative polymerase chain reaction using Taqman® reagents in twenty-four pairs of matched colorectal cancer tumour and associated normal tissue. Differential expression patterns occurred between the normal and malignant tissue and correlated with histopathological parameters and overall surgical staging. The findings demonstrate that a droplet-based microfluidic quantitative PCR system enables biomarker classification. It was further possible to sub-classify colorectal cancer based on extracellular matrix protein expressing groups which in turn correlated with prognosis. PMID:27822332

  11. Tendon extracellular matrix damage, degradation and inflammation in response to in vitro overload exercise

    PubMed Central

    Thorpe, Chavaunne T.; Chaudhry, Saira; Riley, Graham P.; Birch, Helen L.; Clegg, Peter D.; Screen, Hazel R.C.

    2015-01-01

    ABSTRACT The role of inflammation in tendon injury is uncertain and a topic of current interest. In vitro studies of tendon accelerated overload damage can serve as a valuable source of information on the early stages of tendinopathy. Viable fascicle bundles from bovine flexor tendons were subjected to cyclic uniaxial loading from 1–10% strain. Immuno‐staining for inflammatory markers and matrix degradation markers was performed on the samples after mechanical testing. Loaded samples exhibited visible extracellular matrix damage, with disrupted collagen fibers and fiber kinks, and notable damage to the interfascicular matrix. Inflammatory markers COX‐2 and IL‐6 were only expressed in the cyclically loaded samples. Collagen degradation markers MMP‐1 and C1,2C were colocalized in many areas, with staining occurring in the interfascicular matrix or the fascicular tenocytes. These markers were present in control samples, but staining became increasingly intense with loading. Little MMP‐3 or MMP‐13 was evident in control sections. In loaded samples, some sections showed intense staining of these markers, again localized to interfascicular regions. This study suggests that inflammatory markers may be expressed rapidly after tendon overload exercise. Interestingly, both inflammation and damage‐induced matrix remodeling seem to be concentrated in, or in the vicinity of, the highly cellular interfascicular matrix. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 33:889–897, 2015. PMID:25721513

  12. Tendon extracellular matrix damage, degradation and inflammation in response to in vitro overload exercise.

    PubMed

    Spiesz, Ewa M; Thorpe, Chavaunne T; Chaudhry, Saira; Riley, Graham P; Birch, Helen L; Clegg, Peter D; Screen, Hazel R C

    2015-06-01

    The role of inflammation in tendon injury is uncertain and a topic of current interest. In vitro studies of tendon accelerated overload damage can serve as a valuable source of information on the early stages of tendinopathy. Viable fascicle bundles from bovine flexor tendons were subjected to cyclic uniaxial loading from 1-10% strain. Immuno-staining for inflammatory markers and matrix degradation markers was performed on the samples after mechanical testing. Loaded samples exhibited visible extracellular matrix damage, with disrupted collagen fibers and fiber kinks, and notable damage to the interfascicular matrix. Inflammatory markers COX-2 and IL-6 were only expressed in the cyclically loaded samples. Collagen degradation markers MMP-1 and C1,2C were colocalized in many areas, with staining occurring in the interfascicular matrix or the fascicular tenocytes. These markers were present in control samples, but staining became increasingly intense with loading. Little MMP-3 or MMP-13 was evident in control sections. In loaded samples, some sections showed intense staining of these markers, again localized to interfascicular regions. This study suggests that inflammatory markers may be expressed rapidly after tendon overload exercise. Interestingly, both inflammation and damage-induced matrix remodeling seem to be concentrated in, or in the vicinity of, the highly cellular interfascicular matrix. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  13. In Silico Investigation of Angiogenesis with Growth and Stress Generation Coupled to Local Extracellular Matrix Density

    PubMed Central

    Edgar, Lowell T.; Hoying, James B.; Weiss, Jeffrey A.

    2015-01-01

    Mechanical interactions during angiogenesis, i.e., traction applied by neovessels to the extracellular matrix and the corresponding deformation, are important regulators of growth and neovascularization. We have previously designed, implemented, and validated a coupled model of angiogenesis in which a discrete microvessel growth model interacts with a continuous finite element mesh through the application of local remodeling sprout stresses (Edgar et al. in Biomech Model Mechanobiol, 2014). However, the initial implementation of this framework does not take matrix density into account when determined these remodeling stresses and is therefore insufficient for the study of angiogenesis within heterogeneous matrix environments such as those found in vivo. The objective of this study was to implement sensitivity to matrix density in the active stress generation within AngioFE in order to allow the study of angiogenic growth within a heterogeneous density environment. We accomplished this by scaling active sprout stresses relative to local matrix density using a scaling factor previously determined from experimental data. We then exercised the new functionality of the model by simulating angiogenesis within four different scenarios: homogeneous density, a narrow gap model, and matrix density gradient, and a construct subjected to repeated loading/unloading and preconditioning. These numerical experiments predicted heterogeneous matrix density in the initially homogeneous case, the closure and alignment of microvessels along a low-density gap, the formation of a unique cap-like structure during angiogenesis within a density gradient, and the alignment of microvessels in the absence of applied load due to preconditioning. The result of these in silico investigations demonstrate how matrix heterogeneity affects neovascularization and matrix deformation and provides a platform for studying angiogenesis in complicated and multi-faceted mechanical environments that

  14. Modeling the tumor extracellular matrix: Tissue engineering tools repurposed towards new frontiers in cancer biology.

    PubMed

    Gill, Bartley J; West, Jennifer L

    2014-06-27

    Cancer progression is mediated by complex epigenetic, protein and structural influences. Critical among them are the biochemical, mechanical and architectural properties of the extracellular matrix (ECM). In recognition of the ECM's important role, cancer biologists have repurposed matrix mimetic culture systems first widely used by tissue engineers as new tools for in vitro study of tumor models. In this review we discuss the pathological changes in tumor ECM, the limitations of 2D culture on both traditional and polyacrylamide hydrogel surfaces in modeling these characteristics and advances in both naturally derived and synthetic scaffolds to facilitate more complex and controllable 3D cancer cell culture. Studies using naturally derived matrix materials like Matrigel and collagen have produced significant findings related to tumor morphogenesis and matrix invasion in a 3D environment and the mechanotransductive signaling that mediates key tumor-matrix interaction. However, lack of precise experimental control over important matrix factors in these matrices have increasingly led investigators to synthetic and semi-synthetic scaffolds that offer the engineering of specific ECM cues and the potential for more advanced experimental manipulations. Synthetic scaffolds composed of poly(ethylene glycol) (PEG), for example, facilitate highly biocompatible 3D culture, modular bioactive features like cell-mediated matrix degradation and complete independent control over matrix bioactivity and mechanics. Future work in PEG or similar reductionist synthetic matrix systems should enable the study of increasingly complex and dynamic tumor-ECM relationships in the hopes that accurate modeling of these relationships may reveal new cancer therapeutics targeting tumor progression and metastasis. © 2013 Published by Elsevier Ltd.

  15. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System

    PubMed Central

    Chaturvedi, Vishal; Dye, Danielle E.; Kinnear, Beverley F.; van Kuppevelt, Toin H.; Grounds, Miranda D.; Coombe, Deirdre R.

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates. PMID:26030912

  16. Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Langer, R.; Ingber, D. E.

    1995-01-01

    This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In

  17. Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Langer, R.; Ingber, D. E.

    1995-01-01

    This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In

  18. Pilot Study of the Efficacy of Extracellular Matrix Arterial Interposition Grafts in a Sheep (Ovis aries) Model

    DTIC Science & Technology

    2014-02-18

    extracellular matrix arterial interposition grafts in a sheep (Ovis aries) model." 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER...carotid arteries in sheep . Methods: Three crossbred sheep were anesthetized, instrumented, and had 10 cm interposition grafts placed in both carotid...was present by four weeks. Conclusion: In this pilot study, the Cormatrix extracellular matrix performed well in a sheep carotid interposition graft

  19. Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia

    PubMed Central

    McKee, Marc D.; Hoac, Betty; Addison, William N.; Barros, Nilana M.T.; Millán, José Luis; Chaussain, Catherine

    2013-01-01

    As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth – where calcium phosphate crystals are deposited and grow within an extracellular matrix – is essential to dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition such that teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibres (Sharpey's fibres) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here with clinical examples given, namely tissue-nonspecific alkaline phosphatase (TNAP) and phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX). Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH), respectively, where levels of local and systemic circulating mineralization determinants are perturbed. In XLH, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating SIBLING proteins such as matrix extracellular phosphoglycoprotein (MEPE) and osteopontin (OPN), and the phosphorylated peptides proteolytically released from them such as the acidic serine- and aspartate-rich motif (ASARM) peptide, may accumulate locally to impair mineralization in this disease. PMID:23931057

  20. The Dynamic Sclera: Extracellular Matrix Remodeling in Normal Ocular Growth and Myopia Development

    PubMed Central

    Harper, Angelica R.; Summers, Jody A.

    2014-01-01

    Myopia is a common ocular condition, characterized by excessive elongation of the ocular globe. The prevalence of myopia continues to increase, particularly among highly educated groups, now exceeding 80% in some groups. In parallel with the increased prevalence of myopia, are increases in associated blinding ocular conditions including glaucoma, retinal detachment and macular degeneration, making myopia a significant global health concern. The elongation of the eye is closely related to the biomechanical properties of the sclera, which in turn are largely dependent on the composition of the scleral extracellular matrix. Therefore an understanding of the cellular and extracellular events involved in the regulation of scleral growth and remodeling during childhood and young adulthood will provide future avenues for the treatment of myopia and its associated ocular complications. PMID:25819458

  1. Extracellular matrix and cell shape: potential control points for inhibition of angiogenesis

    NASA Technical Reports Server (NTRS)

    Ingber, D.

    1991-01-01

    Capillary endothelial (CE) cells require two extracellular signals in order to switch from quiescence to growth and back to differentiation during angiogenesis: soluble angiogenic factors and insoluble extracellular matrix (ECM) molecules. Soluble endothelial mitogens, such as basic fibroblast growth factor (FGF), act over large distances to trigger capillary growth, whereas ECM molecules act locally to modulate cell responsiveness to these soluble cues. Recent studies reveal that ECM molecules regulate CE cell growth and differentiation by modulating cell shape and by activating intracellular chemical signaling pathways inside the cell. Recognition of the importance of ECM and cell shape during capillary morphogenesis has led to the identification of a series of new angiogenesis inhibitors. Elucidation of the molecular mechanism of capillary regulation may result in development of even more potent angiogenesis modulators in the future.

  2. Casting a Net on Dendritic Spines: The Extracellular Matrix and its Receptors

    PubMed Central

    Dansie, Lorraine E.; Ethell, Iryna M.

    2011-01-01

    Dendritic spines are dynamic structures that accommodate the majority of excitatory synapses in the brain and are influenced by extracellular signals from presynaptic neurons, glial cells and the extracellular matrix (ECM). The ECM surrounds dendritic spines and extends into the synaptic cleft, maintaining synapse integrity as well as mediating trans-synaptic communications between neurons. Several scaffolding proteins and glycans that compose the ECM form a lattice-like network, which serves as an attractive ground for various secreted glycoproteins, lectins, growth factors and enzymes. ECM components can control dendritic spines through the interactions with their specific receptors or by influencing the functions of other synaptic proteins. In this review, we focus on ECM components and their receptors that regulate dendritic spine development and plasticity in the normal and diseased brain. PMID:21834084

  3. Extracellular matrix and cell shape: potential control points for inhibition of angiogenesis

    NASA Technical Reports Server (NTRS)

    Ingber, D.

    1991-01-01

    Capillary endothelial (CE) cells require two extracellular signals in order to switch from quiescence to growth and back to differentiation during angiogenesis: soluble angiogenic factors and insoluble extracellular matrix (ECM) molecules. Soluble endothelial mitogens, such as basic fibroblast growth factor (FGF), act over large distances to trigger capillary growth, whereas ECM molecules act locally to modulate cell responsiveness to these soluble cues. Recent studies reveal that ECM molecules regulate CE cell growth and differentiation by modulating cell shape and by activating intracellular chemical signaling pathways inside the cell. Recognition of the importance of ECM and cell shape during capillary morphogenesis has led to the identification of a series of new angiogenesis inhibitors. Elucidation of the molecular mechanism of capillary regulation may result in development of even more potent angiogenesis modulators in the future.

  4. Insider trading: Extracellular matrix proteins and their non-canonical intracellular roles.

    PubMed

    Hellewell, Andrew L; Adams, Josephine C

    2016-01-01

    In metazoans, the extracellular matrix (ECM) provides a dynamic, heterogeneous microenvironment that has important supportive and instructive roles. Although the primary site of action of ECM proteins is extracellular, evidence is emerging for non-canonical intracellular roles. Examples include osteopontin, thrombospondins, IGF-binding protein 3 and biglycan, and relate to roles in transcription, cell-stress responses, autophagy and cancer. These findings pose conceptual problems on how proteins signalled for secretion can be routed to the cytosol or nucleus, or can function in environments with diverse redox, pH and ionic conditions. We review evidence for intracellular locations and functions of ECM proteins, and current knowledge of the mechanisms by which they may enter intracellular compartments. We evaluate the experimental methods that are appropriate to obtain rigorous evidence for intracellular localisation and function. Better insight into this under-researched topic is needed to decipher the complete spectrum of physiological and pathological roles of ECM proteins.

  5. Lib, transcriptionally induced in senile plaque-associated astrocytes, promotes glial migration through extracellular matrix.

    PubMed

    Satoh, Kazuki; Hata, Mitsumi; Shimizu, Tomoko; Yokota, Hiroshi; Akatsu, Hiroyasu; Yamamoto, Takayuki; Kosaka, Kenji; Yamada, Tatsuo

    2005-09-23

    In an effort to identify astrocyte-derived molecules that may be intimately associated with progression of Alzheimer's disease (AD), Lib, a type I transmembrane protein belonging to leucine-rich repeat superfamily, has been identified as a distinctly inducible gene, responsive to beta-amyloid as well as pro-inflammatory cytokines in astrocytes. To evaluate the roles of Lib in AD, we investigated Lib expression in AD brain. In non-AD brain, Lib mRNA has been detected in neurons but not in quiescent astrocytes. On the contrary, in AD brain, Lib mRNA is expressed in activated astrocytes associated with senile plaques, but not expressed in neurons around lesions. Lib-expressing glioma cells displayed promotion of migration ability through reconstituted extracellular matrix and recombinant Lib protein bound to constituents of extracellular matrix. These observations suggest that Lib may contribute to regulation of cell-matrix adhesion interactions with respect to astrocyte recruitment around senile plaques in AD brain.

  6. The extracellular matrix protein WARP is a novel component of a distinct subset of basement membranes.

    PubMed

    Allen, Justin M; Brachvogel, Bent; Farlie, Peter G; Fitzgerald, Jamie; Bateman, John F

    2008-05-01

    WARP is a recently described member of the von Willebrand factor A domain superfamily of extracellular matrix proteins, and is encoded by the Vwa1 gene. We have previously shown that WARP is a multimeric component of the chondrocyte pericellular matrix in articular cartilage and intervertebral disc, where it interacts with the basement membrane heparan sulfate proteoglycan perlecan. However, the tissue-specific expression of WARP in non-cartilaginous tissues and its localization in the extracellular matrix of other perlecan-containing tissues have not been analyzed in detail. To visualize WARP-expressing cells, we generated a reporter gene knock-in mouse by targeted replacement of the Vwa1 gene with beta-galactosidase. Analysis of reporter gene expression and WARP protein localization by immunostaining demonstrates that WARP is a component of a limited number of distinct basement membranes. WARP is expressed in the vasculature of neural tissues and in basement membrane structures of the peripheral nervous system. Furthermore, WARP is also expressed in the apical ectodermal ridge of developing limb buds, and in skeletal and cardiac muscle. These findings are the first evidence for WARP expression in non-cartilaginous tissues, and the identification of WARP as a component of a limited range of specialized basement membranes provides further evidence for the heterogeneous composition of basement membranes between different tissues.

  7. Scaffolding the retina: the interstitial extracellular matrix during rat retinal development.

    PubMed

    Taylor, Linnéa; Arnér, Karin; Engelsberg, Karl; Ghosh, Fredrik

    2015-05-01

    To examine the expression of interstitial extracellular matrix components and their role during retinal development. Fibronectin (FN), collagen IV (Coll IV) and laminin 5 (Lam 5) expression in rat retinas from developmental stages E17 to adult were studied. In addition, PN5 full-thickness retinas were cultured for 7 days with dispase, which selectively cleaves FN and Coll IV, at either 0.5 U/ml or 5.0 U/ml for 3 or 24h. Eyecups and retinal cultures were examined morphologically using hematoxylin and eosin staining and immunohistochemistry. Coll IV, Lam 5 and FN were all transiently expressed in the interstitial matrix of the retinal layers during development. The retinal layers in dispase treated explants was severely disturbed in a dose and time dependent manner. FN, Lam 5 and Coll IV, are present in the interstitial extracellular matrix during rat retinal development. Enzymatic cleavage of FN and Coll IV early in the lamination process disrupts the retinal layers implicating their pivotal role in this process. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Semicarbazide-sensitive amine oxidase and extracellular matrix deposition by smooth-muscle cells

    NASA Technical Reports Server (NTRS)

    Langford, Shannon D.; Trent, Margaret B.; Boor, Paul J.

    2002-01-01

    We have recently reported in vivo disruption of collagen and elastin architecture within blood vessel walls resulting from the selective inhibition of the enzyme semicarbazide-sensitive amine oxidase (SSAO). This study further investigates the effects of SSAO inhibition on extracellular matrix deposition by smooth-muscle cells (SMCs) cultured from neonatal rat hearts. SMCs were characterized, SSAO activity was measured, and soluble and insoluble collagen and elastin in the extracellular matrix (ECM) were quantified. Cultured neonatal rat heart SMC exhibited a monotypic synthetic phenotype that likely represents a myofibroblast. Detectable levels of SSAO activity present throughout 30-d culture peaked at 7-14 d, coinciding with the production of ECM. The addition of enzyme inhibitors and alternate SSAO substrates (benzylamine) produced varied and, in some cases, marked changes in SSAO activity as well as in the composition of mature and soluble matrix components. Similar to our previous in vivo findings, in vitro SSAO inhibition produced aberrations in collagen and elastin deposition by heart SMC. Because changes in SSAO activity are associated with cardiovascular pathologic states, this enzyme may play a protective or modulating role by regulating ECM production during pathologic insult.

  9. The role of extracellular matrix in peripheral nerve regeneration: a wound chamber study.

    PubMed

    Liu, H M

    1992-01-01

    A wound chamber model was used for the study of the interaction between axon, Schwann cell and extracellular matrix during peripheral nerve regeneration. Impermeable silicone tubes, 8 mm long and 1.4 mm in internal diameter were sutured to transected rat sciatic nerve and the contents of the tubes were removed at intervals for chemical, histological, immunocytochemical and electron microscopic studies. There was an initial phase of fluid accumulation and the formation of a fibrin/fibronectin clot or cable which connected the cut ends of the nerve. The chamber fluid was shown to have a protein profile similar to that of rat serum. Schwann cells, endothelial cells and fibroblasts migrated first into the cable, apparently mediated by cell-fibrin interaction. Axons buried within the Schwann cell cytoplasm were led into the cable but an axon-fibrin interaction was not observed. After 1 week, the fibrin matrix underwent dissolution, with replacement by collagen. This marked the onset of myelination and the organization of nerve fibers into fascicles. The findings from the present study suggest that the interactions between axon and Schwann cell and between Schwann cell and a changing extracellular matrix are the essential driving force in nerve growth and differentiation during peripheral nerve regeneration.

  10. Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

    PubMed Central

    Zhang, Wei; Chen, Xiao-Ping; Zhang, Wan-Guang; Zhang, Feng; Xiang, Shuai; Dong, Han-Hua; Zhang, Lei

    2009-01-01

    AIM: To elucidate the interaction between non-parenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover. CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions. PMID:19195056

  11. Semicarbazide-sensitive amine oxidase and extracellular matrix deposition by smooth-muscle cells

    NASA Technical Reports Server (NTRS)

    Langford, Shannon D.; Trent, Margaret B.; Boor, Paul J.

    2002-01-01

    We have recently reported in vivo disruption of collagen and elastin architecture within blood vessel walls resulting from the selective inhibition of the enzyme semicarbazide-sensitive amine oxidase (SSAO). This study further investigates the effects of SSAO inhibition on extracellular matrix deposition by smooth-muscle cells (SMCs) cultured from neonatal rat hearts. SMCs were characterized, SSAO activity was measured, and soluble and insoluble collagen and elastin in the extracellular matrix (ECM) were quantified. Cultured neonatal rat heart SMC exhibited a monotypic synthetic phenotype that likely represents a myofibroblast. Detectable levels of SSAO activity present throughout 30-d culture peaked at 7-14 d, coinciding with the production of ECM. The addition of enzyme inhibitors and alternate SSAO substrates (benzylamine) produced varied and, in some cases, marked changes in SSAO activity as well as in the composition of mature and soluble matrix components. Similar to our previous in vivo findings, in vitro SSAO inhibition produced aberrations in collagen and elastin deposition by heart SMC. Because changes in SSAO activity are associated with cardiovascular pathologic states, this enzyme may play a protective or modulating role by regulating ECM production during pathologic insult.

  12. Carboxymethylcellulose hydrogels support central nervous system-derived tumor-cell chemotactic migration: comparison with conventional extracellular matrix macromolecules.

    PubMed

    Singh, Tanya; Kothapalli, Chandrasekhar; Varma, Devika; Nicoll, Steven B; Vazquez, Maribel

    2014-09-01

    The local microenvironment plays an important role in maintaining the dynamics of the extracellular matrix and the cell-extracellular matrix relationship. The extracellular matrix is a complex network of macromolecules with distinct mechanical and biochemical characteristics. Disruptions in extracellular matrix homeostasis are associated with the onset of cancer. The extracellular matrix becomes highly disorganized, and the cell-matrix relationship changes, resulting in altered cell-signaling processes and metastasis. Medulloblastoma is one of the most common malignant pediatric brain tumors in the United States. In order to gain a better understanding of the interplay between cell-extracellular matrix interactions and cell-migratory responses in tumors, eight different matrix macromolecule formulations were investigated using a medulloblastoma-derived cell line: poly-D-lysine, matrigel, laminin, collagen 1, fibronectin, a 10% blend of laminin-collagen 1, a 20% blend of laminin-collagen 1, and a cellulose-derived hydrogel, carboxymethylcellulose. Over time, the average changes in cell morphology were quantified in 2D and 3D, as was migration in the presence and absence of the chemoattractant, epidermal growth factor. Data revealed that carboxymethylcellulose allowed for a cell-extracellular matrix relationship typically believed to be present in tumors, with cells exhibiting a rounded, amoeboid morphology consistent with chemotactic migration, while the other matrices promoted an elongated cell shape as well as both haptotactic and chemotactic motile processes. Therefore, carboxymethylcellulose hydrogels may serve as effective platforms for investigating central nervous system-derived tumor-cell migration in response to soluble factors. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  13. The fiber diameter of synthetic bioresorbable extracellular matrix influences human fibroblast morphology and fibronectin matrix assembly.

    PubMed

    Hsia, Henry C; Nair, Mohan R; Mintz, R Candida; Corbett, Siobhan A

    2011-06-01

    The ideal scaffold material should provide immediate capacity to bear mechanical loads and also permit eventual resorption and replacement with native tissue of similar mechanical integrity. Scaffold characteristics such as fiber diameter provide environmental cues that can influence cell function and differentiation. In this study, the impact of fiber diameter of scaffolds constructed from a tyrosine-based bioresorbable polymer on cellular response was investigated. Electrospun bioresorbable poly(desamino tyrosyl-tyrosine ethyl ester carbonate) scaffolds composed of microfibers or nanofibers were constructed and seeded with human dermal fibroblasts. The impact of fiber diameter on actin cytoskeletal morphology, focal adhesion size, fibronectin matrix assembly, and cell proliferation was evaluated using immunofluorescent microscopy and computer-assisted image analysis. Actin stress fibers were more easily observed in cells on microfiber scaffolds compared with those on nanofiber scaffolds. Cells on nanofiber scaffolds developed smaller focal adhesion complexes compared with those on microfiber scaffolds (p < 0.0001). The temporal patterns of fibronectin matrix assembly were affected by scaffold fiber diameter, with cells on microfiber scaffolds showing a delayed response in dense fibril formation compared with nanofiber scaffolds. Cells on nanofiber scaffolds showed higher proliferation compared with microfiber scaffolds at time points under 1 week (p < 0.01), but by 2 weeks significantly higher cell proliferation was observed on microfiber scaffolds (p < 0.01). The fiber diameter of bioresorbable scaffolds can significantly influence cell response and suggests that the ability of scaffolds to elicit consistent biological responses depends on factors beyond scaffold composition. Such findings have important implications for the design of clinically useful engineered constructs.

  14. Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold.

    PubMed

    Kelly, Brian A; Proffen, Benedikt L; Haslauer, Carla M; Murray, Martha M

    2016-04-01

    The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  15. The contribution of the extracellular matrix to gravisensing in characean cells

    NASA Technical Reports Server (NTRS)

    Wayne, R.; Staves, M. P.; Leopold, A. C.

    1992-01-01

    The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

  16. Extracellular matrix assembly in extreme acidic eukaryotic biofilms and their possible implications in heavy metal adsorption.

    PubMed

    Aguilera, Angeles; Souza-Egipsy, Virginia; Martín-Uriz, Patxi San; Amils, Ricardo

    2008-07-30

    To evaluate the importance of the extracellular matrix in relation to heavy metal binding capacity in extreme acidic environments, the extracellular polymeric substances (EPS) composition of 12 biofilms isolated from Río Tinto (SW, Spain) was analyzed. Each biofilm was composed mainly by one or two species of eukaryotes, although other microorganisms were present. EPS ranged from 130 to 439 mg g(-1) biofilm dry weight, representing between 15% and the 40% of the total biofilm dry weight (DW). Statistically significant differences (p<0.05) were found in the amount of total EPS extracted from biofilms dominated by the same organism at different sampling points. The amount of EPS varied among different biofilms collected from the same sampling location. Colloidal EPS ranged from 42 to 313 mg g(-1) dry weight; 10% to 30% of the total biofilm dry weight. Capsular EPS ranged from 50 to 318 mg g(-1) dry weight; 5% to 30% of the total biofilm dry weight. Seven of the 12 biofilms showed higher amounts of capsular than colloidal EPS (p<0.05). Total amount of EPS decreased when total cell numbers and pH increased. There was a positive correlation between EPS concentration and heavy metal concentration in the water. Observations by low temperature scanning electron microscopy (LTSEM) revealed the mineral adsorption in the matrix of EPS and onto the cell walls. EPS in all biofilms were primarily composed of carbohydrates, heavy metals and humic acid, plus small quantities of proteins and DNA. After carbohydrates, heavy metals were the second main constituents of the extracellular matrix. Their total concentrations ranged from 3 to 32 mg g(-1) biofilm dry weight, reaching up to 16% of the total composition. In general, the heavy metal composition of the EPS extracted from the biofilms closely resembled the metal composition of the water from which the biofilms were collected.

  17. Gonadotropin-releasing hormone analogues inhibit leiomyoma extracellular matrix despite presence of gonadal hormones.

    PubMed

    Malik, Minnie; Britten, Joy; Cox, Jeris; Patel, Amrita; Catherino, William H

    2016-01-01

    To determine the effect of GnRH analogues (GnRH-a) leuprolide acetate (LA) and cetrorelix acetate on gonadal hormone-regulated expression of extracellular matrix in uterine leiomyoma three-dimensional (3D) cultures. Laboratory study. University research laboratory. Women undergoing hysterectomy for symptomatic leiomyomas. The 3D cell cultures, protein analysis, Western blot, immunohistochemistry. Expression of extracellular matrix proteins, collagen 1, fibronectin, and versican in leiomyoma cells 3D cultures exposed to E2, P, LA, cetrorelix acetate, and combinations for 24- and 72-hour time points. The 3D leiomyoma cultures exposed to E2 for 24 hours demonstrated an increased expression of collagen-1 and fibronectin, which was maintained for up to 72 hours, a time point at which versican was up-regulated significantly. Although P up-regulated collagen-1 protein (1.29 ± 0.04) within 24 hours of exposure, significant increase in all extracellular matrix (ECM) proteins was observed when the gonadal hormones were used concomitantly. Significant decrease in the amount of ECM proteins was observed on use of GnRH-a, LA and cetrorelix, with 24-hour exposure. Both the compounds also significantly decreased ECM protein concentration despite the presence of E2 or both gonadal hormones. This study demonstrates that GnRH-a directly affect the gonadal hormone-regulated collagen-1, fibronectin, and versican production in their presence. These findings suggest that localized therapy with GnRH-a may inhibit leiomyoma growth even in the presence of endogenous gonadal hormone exposure, thereby providing a mechanism to eliminate the hypoestrogenic side effects associated with GnRH-a therapy. Published by Elsevier Inc.

  18. The contribution of the extracellular matrix to gravisensing in characean cells

    NASA Technical Reports Server (NTRS)

    Wayne, R.; Staves, M. P.; Leopold, A. C.

    1992-01-01

    The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

  19. Cadherin-11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves.

    PubMed

    Sung, Derek C; Bowen, Caitlin J; Vaidya, Kiran A; Zhou, Jingjing; Chapurin, Nikita; Recknagel, Andrew; Zhou, Bin; Chen, Jonathan; Kotlikoff, Michael; Butcher, Jonathan T

    2016-08-01

    Calcific aortic valve (AoV) disease is a significant clinical problem for which the regulatory mechanisms are poorly understood. Enhanced cell-cell adhesion is a common mechanism of cellular aggregation, but its role in calcific lesion formation is not known. Cadherin-11 (Cad-11) has been associated with lesion formation in vitro, but its function during adult valve homeostasis and pathogenesis is not known. This study aims to elucidate the specific functions of Cad-11 and its downstream targets, RhoA and Sox9, in extracellular matrix remodeling and AoV calcification. We conditionally overexpressed Cad-11 in murine heart valves using a novel double-transgenic Nfatc1(Cre);R26-Cad11(TglTg) mouse model. These mice developed hemodynamically significant aortic stenosis with prominent calcific lesions in the AoV leaflets. Cad-11 overexpression upregulated downstream targets, RhoA and Sox9, in the valve interstitial cells, causing calcification and extensive pathogenic extracellular matrix remodeling. AoV interstitial cells overexpressing Cad-11 in an osteogenic environment in vitro rapidly form calcific nodules analogous to in vivo lesions. Molecular analyses revealed upregulation of osteoblastic and myofibroblastic markers. Treatment with a Rho-associated protein kinase inhibitor attenuated nodule formation, further supporting that Cad-11-driven calcification acts through the small GTPase RhoA/Rho-associated protein kinase signaling pathway. This study identifies one of the underlying molecular mechanisms of heart valve calcification and demonstrates that overexpression of Cad-11 upregulates RhoA and Sox9 to induce calcification and extracellular matrix remodeling in adult AoV pathogenesis. The findings provide a potential molecular target for clinical treatment. © 2016 American Heart Association, Inc.

  20. Local extracellular matrix alignment directs cellular protrusion dynamics and migration through Rac1 and FAK.

    PubMed

    Carey, Shawn P; Goldblatt, Zachary E; Martin, Karen E; Romero, Bethsabe; Williams, Rebecca M; Reinhart-King, Cynthia A

    2016-08-08

    Cell migration within 3D interstitial microenvironments is sensitive to extracellular matrix (ECM) properties, but the mechanisms that regulate migration guidance by 3D matrix features remain unclear. To examine the mechanisms underlying the cell migration response to aligned ECM, which is prevalent at the tumor-stroma interface, we utilized time-lapse microscopy to compare the behavior of MDA-MB-231 breast adenocarcinoma cells within randomly organized and well-aligned 3D collagen ECM. We developed a novel experimental system in which cellular morphodynamics during initial 3D cell spreading served as a reductionist model for the complex process of matrix-directed 3D cell migration. Using this approach, we found that ECM alignment induced spatial anisotropy of cells' matrix probing by promoting protrusion frequency, persistence, and lengthening along the alignment axis and suppressing protrusion dynamics orthogonal to alignment. Preference for on-axis behaviors was dependent upon FAK and Rac1 signaling and translated across length and time scales such that cells within aligned ECM exhibited accelerated elongation, front-rear polarization, and migration relative to cells in random ECM. Together, these findings indicate that adhesive and protrusive signaling allow cells to respond to coordinated physical cues in the ECM, promoting migration efficiency and cell migration guidance by 3D matrix structure.

  1. Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching

    PubMed Central

    Plenge, Amelie; Heller, Sandra; Pfeiffer, Bastian; Kasper, Cornelia

    2016-01-01

    Tendon and ligament pathologies are still a therapeutic challenge, due to the difficulty in restoring the complex extracellular matrix architecture and biomechanical strength. While progress is being made in cell-based therapies and tissue engineering approaches, comprehensive understanding of the fate of progenitor cells in tendon healing is still lacking. The aim of this study was to investigate the effect of decellularized tendon matrix and moderate cyclic stretching as natural stimuli which could potentially direct tenogenic fate. Equine adipose-derived mesenchymal stromal cells (MSC) were seeded on decellularized tendon matrix scaffolds. Mechanical stimulation was applied in a custom-made cyclic strain bioreactor. Assessment was performed 4 h, 8 h, and 24 h following mechanical stimulation. Scaffold culture induced cell alignment and changes in expression of tendon-related genes, although cell viability was decreased compared to monolayer culture. Short mechanical stimulation periods enhanced most of the scaffold-induced effects. Collagen 1A2 expression levels were decreased, while collagen 3A1 and decorin levels were increased. Tenascin-C and scleraxis expression showed an initial decrease but had increased 24 h after stimulation. The results obtained suggest that decellularized tendon matrix, supported by cyclic stretching, can induce tenogenic differentiation and the synthesis of tendon components important for matrix remodeling. PMID:27630718

  2. Teaching the Extracellular Matrix and Introducing Online Databases within a Multidisciplinary Course with i-Cell-MATRIX: A Student-Centered Approach

    ERIC Educational Resources Information Center

    Sousa, Joao Carlos; Costa, Manuel Joao; Palha, Joana Almeida

    2010-01-01

    The biochemistry and molecular biology of the extracellular matrix (ECM) is difficult to convey to students in a classroom setting in ways that capture their interest. The understanding of the matrix's roles in physiological and pathological conditions study will presumably be hampered by insufficient knowledge of its molecular structure.…

  3. Teaching the Extracellular Matrix and Introducing Online Databases within a Multidisciplinary Course with i-Cell-MATRIX: A Student-Centered Approach

    ERIC Educational Resources Information Center

    Sousa, Joao Carlos; Costa, Manuel Joao; Palha, Joana Almeida

    2010-01-01

    The biochemistry and molecular biology of the extracellular matrix (ECM) is difficult to convey to students in a classroom setting in ways that capture their interest. The understanding of the matrix's roles in physiological and pathological conditions study will presumably be hampered by insufficient knowledge of its molecular structure.…

  4. Adhesion molecules and the extracellular matrix as drug targets for glioma.

    PubMed

    Shimizu, Toshihiko; Kurozumi, Kazuhiko; Ishida, Joji; Ichikawa, Tomotsugu; Date, Isao

    2016-04-01

    The formation of tumor vasculature and cell invasion along white matter tracts have pivotal roles in the development and progression of glioma. A better understanding of the mechanisms of angiogenesis and invasion in glioma will aid the development of novel therapeutic strategies. The processes of angiogenesis and invasion cause the production of an array of adhesion molecules and extracellular matrix (ECM) components. This review focuses on the role of adhesion molecules and the ECM in malignant glioma. The results of clinical trials using drugs targeted against adhesion molecules and the ECM for glioma are also discussed.

  5. Of extracellular matrix, scaffolds, and signaling: Tissuearchitectureregulates development, homeostasis, and cancer

    SciTech Connect

    Nelson, Celeste M.; Bissell, Mina J.

    2006-03-09

    The microenvironment surrounding cells influences gene expression, such that a cell's behavior is largely determined by its interactions with the extracellular matrix, neighboring cells, and soluble cues released locally or by distant tissues. We describe the essential role of context and organ structure in directing mammary gland development and differentiated function, and in determining response to oncogenic insults including mutations. We expand on the concept of 'dynamic reciprocity' to present an integrated view of development, cancer, and aging, and posit that genes are like piano keys: while essential, it is the context that makes the music.

  6. Possible Involvement of Tight Junctions, Extracellular Matrix and Nuclear Receptors in Epithelial Differentiation

    PubMed Central

    Ichikawa-Tomikawa, Naoki; Sugimoto, Kotaro; Satohisa, Seiro; Nishiura, Keisuke; Chiba, Hideki

    2011-01-01

    Tight junctions are intercellular junctions localized at the most apical end of the lateral plasma membrane. They consist of four kinds of transmembrane proteins (occludin, claudins, junctional adhesion molecules, and tricellulin) and huge numbers of scaffolding proteins and contribute to the paracellular barrier and fence function. The mutation and deletion of these proteins impair the functions of tight junctions and cause various human diseases. In this paper, we provide an overview of recent studies on transmembrane proteins of tight junctions and highlight the functional significance of tight junctions, extracellular matrix, and nuclear receptors in epithelial differentiation. PMID:22162632

  7. Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

    SciTech Connect

    Boudreau, Nancy; Sympson, C. J.; Werb, Zena; Bissell, Mina J.

    1994-12-01

    Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.

  8. Emerging Implications for Extracellular Matrix-Based Technologies in Vascularized Composite Allotransplantation

    PubMed Central

    Londono, Ricardo; Gorantla, Vijay S.; Badylak, Stephen F.

    2016-01-01

    Despite recent progress in vascularized composite allotransplantation (VCA), limitations including complex, high dose immunosuppression regimens, lifelong risk of toxicity from immunosuppressants, acute and most critically chronic graft rejection, and suboptimal nerve regeneration remain particularly challenging obstacles restricting clinical progress. When properly configured, customized, and implemented, biomaterials derived from the extracellular matrix (ECM) retain bioactive molecules and immunomodulatory properties that can promote stem cell migration, proliferation and differentiation, and constructive functional tissue remodeling. The present paper reviews the emerging implications of ECM-based technologies in VCA, including local immunomodulation, tissue repair, nerve regeneration, minimally invasive graft targeted drug delivery, stem cell transplantation, and other donor graft manipulation. PMID:26839554

  9. The Extracellular Matrix in Epithelial Ovarian Cancer – A Piece of a Puzzle

    PubMed Central

    Cho, Angela; Howell, Viive M.; Colvin, Emily K.

    2015-01-01

    Epithelial ovarian cancer is the fifth leading cause of cancer-related deaths in women and the most lethal gynecological malignancy. Extracellular matrix (ECM) is an integral component of both the normal and tumor microenvironment. ECM composition varies between tissues and is crucial for maintaining normal function and homeostasis. Dysregulation and aberrant deposition or loss of ECM components is implicated in ovarian cancer progression. The mechanisms by which tumor cells induce ECM remodeling to promote a malignant phenotype are yet to be elucidated. A thorough understanding of the role of the ECM in ovarian cancer is needed for the development of effective biomarkers and new therapies. PMID:26579497

  10. Back to basics--how the evolution of the extracellular matrix underpinned vertebrate evolution.

    PubMed

    Huxley-Jones, Julie; Pinney, John W; Archer, John; Robertson, David L; Boot-Handford, Raymond P

    2009-04-01

    The extracellular matrix (ECM) is a complex substrate that is involved in and influences a spectrum of behaviours such as growth and differentiation and is the basis for the structure of tissues. Although a characteristic of all metazoans, the ECM has elaborated into a variety of tissues unique to vertebrates, such as bone, tendon and cartilage. Here we review recent advances in our understanding of the molecular evolution of the ECM. Furthermore, we demonstrate that ECM genes represent a pivotal family of proteins the evolution of which appears to have played an important role in the evolution of vertebrates.

  11. Local Microarchitecture Affects Mechanical Properties of Deposited Extracellular Matrix for Osteonal Regeneration

    DTIC Science & Technology

    2013-10-31

    Aguero, M.R. Appleford Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX, USA a b s t r a c ta r t i c l e i...Deposited Extracellular Matrix for Osteonal Regeneration 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR( S ) Pilia M., Pollot B...E., Aguero V., Appleford M. R., 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME( S ) AND ADDRESS(ES

  12. "Click" immobilization of a VEGF-mimetic peptide on decellularized endothelial extracellular matrix to enhance angiogenesis.

    PubMed

    Wang, Lin; Zhao, Meirong; Li, Siheng; Erasquin, Uriel J; Wang, Hao; Ren, Li; Chen, Changyi; Wang, Yingjun; Cai, Chengzhi

    2014-06-11

    We show that coating of decellularized extracellular matrix (DC-ECM) on substrate surfaces is an efficient way to generate a platform mimicking the native ECM environment. Moreover, the DC-ECM can be modified with a peptide (QK) mimicking vascular endothelial growth factor without apparently compromising its integrity. The modification was achieved through metabolic incorporation of a "clickable" handle to DC-ECM followed by rapid attachment of the QK peptide with an azido tag using copper-catalyzed click reaction. The attachment of the QK peptide on to DC-ECM in this way further enhanced the angiogenic responses (formation of branched tubular networks) of endothelial cells.

  13. In vitro enhancement of extracellular matrix formation as natural bioscaffold for stem cell culture

    NASA Astrophysics Data System (ADS)

    Naroeni, Aroem; Shalihah, Qonitha; Meilany, Sofy

    2017-02-01

    Growing cells in plastic with liquid media for in vitro study is very common but far from physiological. The use of scaffold materials is more biocompatible. Extracellular matrix provides tissue integrity which acts as a native scaffold for cell attachment and interaction, as well as it serves as a reservoir for growth factors. For this reason, we have developed natural scaffold from mice fibroblast to form a natural scaffold for stem cell culture. Fibroblasts were cultured under crowded condition and lysed to form natural scaffold. The natural scaffold formation was observed using immunofluorescence which then will be used and tested for stem cell propagation and differentiation.

  14. Biomechanical analysis of rotator cuff repairs with extracellular matrix graft augmentation.

    PubMed

    Ely, Erin E; Figueroa, Nathania M; Gilot, Gregory J

    2014-09-01

    Despite advances in surgical techniques, 20% to 90% of rotator cuff (RTC) repairs fail. They tend to fail at the suture-tendon junction due to tension at the repair and gap formation prior to healing. This study evaluated the gap formation and ultimate tensile failure loads of a RTC repair with a decellularized human dermal allograft. Augmentation of a RTC repair with an extracellular matrix graft decreased gap formation and increased load to failure in a human RTC repair model. Copyright 2014, SLACK Incorporated.

  15. Enzymes in the extracellular matrix of Volvox: an inducible, calcium-dependent phosphatase with a modular composition.

    PubMed

    Hallmann, A

    1999-01-15

    The volvocine algae provide the unique opportunity for exploring development of an extracellular matrix. Volvox is the most advanced member of this family and represents the simplest multicellular organism, with differentiated cells, a complete division of labor, and a complex extracellular matrix, which serves structural and enzymatic functions. In Volvox carteri a glycosylated extracellular phosphatase was identified, which is partially released from the extracellular matrix into the growth medium. The phosphatase is synthesized in response to inorganic phosphate starvation and is strictly calcium-dependent. The metalloenzyme has been purified to homogeneity and characterized. Its gene and cDNA have been cloned. Comparisons of genomic and cDNA sequences revealed an extremely intron-rich gene (32 introns). With an apparent molecular mass of 160 kDa the Volvox extracellular phosphatase is the largest phosphatase cloned, with no sequence similarity to any other phosphatase. This enzyme exhibits a modular composition. There are two large domains and a small one. The large domains are highly homologous to each other and therefore most likely originated from gene duplication and fusion. At least one EF-hand motif for calcium binding was identified in this extracellular protein. Volvox extracellular phosphatase is the first calcium-dependent extracellular phosphatase to be cloned.

  16. Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism

    SciTech Connect

    Cooper, D.N.; Barondes, S.H. )

    1990-05-01

    A soluble lactose-binding lectin with subunit Mr of 14,500 is believed to function by interacting with extracellular glycoconjugates, because it has been detected extracellularly by immunohistochemistry. This localization has been questioned, however, since the lectin lacks a secretion signal sequence, which challenges the contention that it is secreted. We have demonstrated externalization of this lectin from C2 mouse muscle cells by both immunoprecipitation of metabolically labeled protein and immunohistochemical localization. We further show that externalization of the lectin is a developmentally regulated process that accompanies myoblast differentiation and that the lectin codistributes with laminin in myotube extracellular matrix. Immunohistochemical localization during intermediate stages of externalization suggests that the lectin becomes concentrated in evaginations of plasma membrane, which pinch off to form labile lectin-rich extracellular vesicles. This suggests a possible mechanism for lectin export from the cytosol to the extracellular matrix.

  17. Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism

    PubMed Central

    1990-01-01

    A soluble lactose-binding lectin with subunit Mr of 14,500 is believed to function by interacting with extracellular glycoconjugates, because it has been detected extracellularly by immunohistochemistry. This localization has been questioned, however, since the lectin lacks a secretion signal sequence, which challenges the contention that it is secreted. We have demonstrated externalization of this lectin from C2 mouse muscle cells by both immunoprecipitation of metabolically labeled protein and immunohistochemical localization. We further show that externalization of the lectin is a developmentally regulated process that accompanies myoblast differentiation and that the lectin codistributes with laminin in myotube extracellular matrix. Immunohistochemical localization during intermediate stages of externalization suggests that the lectin becomes concentrated in evaginations of plasma membrane, which pinch off to form labile lectin- rich extracellular vesicles. This suggests a possible mechanism for lectin export from the cytosol to the extracellular matrix. PMID:2335567

  18. Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix.

    PubMed

    Absher, M; Baldor, L

    1991-01-01

    The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells.

  19. Quantitative analysis of 3D extracellular matrix remodelling by pancreatic stellate cells

    PubMed Central

    Robinson, Benjamin K.; Cortes, Ernesto; Rice, Alistair J.; Sarper, Muge

    2016-01-01

    ABSTRACT Extracellular matrix (ECM) remodelling is integral to numerous physiological and pathological processes in biology, such as embryogenesis, wound healing, fibrosis and cancer. Until recently, most cellular studies have been conducted on 2D environments where mechanical cues significantly differ from physiologically relevant 3D environments, impacting cellular behaviour and masking the interpretation of cellular function in health and disease. We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling. Monitoring and quantification of collagen-I structure in remodelled matrices, through designated algorithms, show that 3D matrices can be used to correlate remodelling with increased ECM stiffness observed in fibrosis. Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer. We use PSCs to implement our methodology and demonstrate that PSC matrix remodelling capabilities depend on their contractile machinery and β1 integrin-mediated cell-ECM attachment. PMID:27170254

  20. Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix

    PubMed Central

    Subramanian, Arul; Schilling, Thomas F.

    2015-01-01

    Tendons and ligaments are extracellular matrix (ECM)-rich structures that interconnect muscles and bones. Recent work has shown how tendon fibroblasts (tenocytes) interact with muscles via the ECM to establish connectivity and strengthen attachments under tension. Similarly, ECM-dependent interactions between tenocytes and cartilage/bone ensure that tendon-bone attachments form with the appropriate strength for the force required. Recent studies have also established a close lineal relationship between tenocytes and skeletal progenitors, highlighting the fact that defects in signals modulated by the ECM can alter the balance between these fates, as occurs in calcifying tendinopathies associated with aging. The dynamic fine-tuning of tendon ECM composition and assembly thus gives rise to the remarkable characteristics of this unique tissue type. Here, we provide an overview of the functions of the ECM in tendon formation and maturation that attempts to integrate findings from developmental genetics with those of matrix biology. PMID:26672092

  1. Synthetic Mimics of the Extracellular Matrix: How Simple is Complex Enough?

    PubMed Central

    Kyburz, Kyle A.; Anseth, Kristi S.

    2015-01-01

    Cells reside in a complex and dynamic extracellular matrix where they interact with a myriad of biophysical and biochemical cues that direct their function and regulate tissue homeostasis, wound repair, and even pathophysiological events. There is a desire in the biomaterials community to develop synthetic hydrogels to recapitulate facets of the ECM for in vitro culture platforms and tissue engineering applications. Advances in synthetic hydrogel design and chemistries, including user-tunable platforms, have broadened the field’s understanding of the role of matrix cues in directing cellular processes and enabled the design of improved tissue engineering scaffolds. This review focuses on recent advances in the development and fabrication of hydrogels and discusses what aspects of ECM signals can be incorporated to direct cell function in different contexts. PMID:25753017

  2. [Theodor Huzella and the initiation of research on the interactions between cells and the extracellular matrix].

    PubMed

    Robert, Ladislas; Labat-Robert, Jacqueline; Michel Robert, Alexandre

    2012-01-01

    Interactions between cells and the surrounding "biomatrix", mediated by receptors as integrins or the elastin receptor is the most important topic in up to date research on connective tissues. Looking for the origin of this concept, one finds the pioneering work of Theodor Huzella, professor of histology-embryology at the Medical University of Budapest during pre-world war II decades. Using time-laps micro-cinematography in reflected light, he visualized the important role of connective tissue fibers, prepared in his laboratory, for the oriented migration of normal and malignant cells. His theoretical explanations, attributing an "active" role to the elasticity of the argyrophilic fibrous network in the coordination of cell societies, can now be reinterpreted in the light of recent work on the mechanotransduction of "messages" from the extracellular matrix to the cell inside. We propose a succinct review of Huzella's work and theories reinterpreted in the light of up-to-date knowledge on cell-matrix interactions.

  3. Probing Micromechanical Properties of the Extracellular Matrix of Soft Tissues by Atomic Force Microscopy.

    PubMed

    Jorba, Ignasi; Uriarte, Juan J; Campillo, Noelia; Farré, Ramon; Navajas, Daniel

    2017-01-01

    The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy (AFM) has unique capabilities to measure ECM mechanics at the scale at which cells probe the mechanical features of their microenvironment. Moreover, AFM measurements can be readily combined with bright field and fluorescence microscopy. Performing reliable mechanical measurements with AFM requires accurate calibration of the device and correct computation of the mechanical parameters. A suitable approach to isolate ECM mechanics from cell contribution is removing the cells by means of an effective decellularization process that preserves the composition, structure and mechanical properties of the ECM. AFM measurement of ECM micromechanics provides important insights into organ biofabrication, cell-matrix mechanical crosstalk and disease-induced tissue stiffness alterations. J. Cell. Physiol. 232: 19-26, 2017. © 2016 Wiley Periodicals, Inc.

  4. Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

    NASA Technical Reports Server (NTRS)

    Spooner, B. S.; Bassett, K. E.; Spooner, B. S. Jr

    1993-01-01

    The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.

  5. Systems biology—opportunities and challenges: the application of proteomics to study the cardiovascular extracellular matrix

    PubMed Central

    Barallobre-Barreiro, Javier; Lynch, Marc; Yin, Xiaoke; Mayr, Manuel

    2016-01-01

    Systems biology approaches including proteomics are becoming more widely used in cardiovascular research. In this review article, we focus on the application of proteomics to the cardiac extracellular matrix (ECM). ECM remodelling is a hallmark of many cardiovascular diseases. Proteomic techniques using mass spectrometry (MS) provide a platform for the comprehensive analysis of ECM proteins without a priori assumptions. Proteomics overcomes various constraints inherent to conventional antibody detection. On the other hand, studies that use whole tissue lysates for proteomic analysis mask the identification of the less abundant ECM constituents. In this review, we first discuss decellularization-based methods that enrich for ECM proteins in cardiac tissue, and how targeted MS allows for accurate protein quantification. The second part of the review will focus on post-translational modifications including hydroxylation and glycosylation and on the release of matrix fragments with biological activity (matrikines), all of which can be interrogated by proteomic techniques. PMID:27635058

  6. Development of biomimetic nanocomposites as bone extracellular matrix for human osteoblastic cells.

    PubMed

    Bhowmick, Arundhati; Mitra, Tapas; Gnanamani, Arumugam; Das, Manas; Kundu, Patit Paban

    2016-05-05

    Here, we have developed biomimetic nanocomposites containing chitosan, poly(vinyl alcohol) and nano-hydroxyapatite-zinc oxide as bone extracellular matrix for human osteoblastic cells and characterized by Fourier transform infrared spectroscopy, powder X-ray diffraction. Scanning electron microscopy images revealed interconnected macroporous structures. Moreover, in this study, the problem related to fabricating a porous composite with good mechanical strength has been resolved by incorporating 5wt% of nano-hydroxyapatite-zinc oxide into chitosan-poly(vinyl alcohol) matrix; the present composite showed high tensile strength (20.25MPa) while maintaining appreciable porosity (65.25%). These values are similar to human cancellous bone. These nanocomposites also showed superior water uptake, antimicrobial and biodegradable properties than the previously reported results. Compatibility with human blood and pH was observed, indicating nontoxicity of these materials to the human body. Moreover, proliferation of osteoblastic MG-63 cells onto the nanocomposites was also observed without having any negative effect.

  7. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Ovijit; Koshy, Sandeep T.; Branco da Cunha, Cristiana; Shin, Jae-Won; Verbeke, Catia S.; Allison, Kimberly H.; Mooney, David J.

    2014-10-01

    In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes.

  8. Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

    PubMed

    Simpson, D G; Terracio, L; Terracio, M; Price, R L; Turner, D C; Borg, T K

    1994-10-01

    Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

  9. Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures.

    PubMed

    Stock, U A; Wiederschain, D; Kilroy, S M; Shum-Tim, D; Khalil, P N; Vacanti, J P; Mayer, J E; Moses, M A

    2001-03-26

    Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial requirements for their long-term patency and function. This complex process requires the deposition and accumulation of extracellular matrix molecules as well as the remodeling of this extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). In this study, we have investigated the dynamics of ECM production and the activity of MMPs and TIMPs in long-term tissue-engineered vascular conduits using quantitative ECM analysis, substrate gel electrophoresis, radiometric enzyme assays and Western blot analyses. Over a time period of 169 days in vivo, levels of elastin and proteoglycans/glycosaminoglycans in tissue-engineered constructs came to approximate those of their native tissue counter parts. The kinetics of collagen deposition and remodeling, however, apparently require a much longer time period. Through the use of substrate gel electrophoresis, proteolytic bands whose molecular weight was consistent with their identification as the active form of MMP-2 (approximately 64--66 kDa) were detected in all native and tissue-engineered samples. Additional proteolytic bands migrating at approximately 72 kDa representing the latent form of MMP-2 were detected in tissue-engineered samples at time points from 5 throughout 55 days. Radiometric assays of MMP-1 activity demonstrated no significant differences between the native and tissue-engineered samples. This study determines the dynamics of ECM production and turnover in a long-term tissue-engineered vascular tissue and highlights the importance of ECM remodeling in the development of successful tissue-engineered vascular structures.

  10. Matrix Rigidity Induces Osteolytic Gene Expression of Metastatic Breast Cancer Cells

    PubMed Central

    Ruppender, Nazanin S.; Merkel, Alyssa R.; Martin, T. John; Sterling, Julie A.; Guelcher, Scott A.

    2010-01-01

    Nearly 70% of breast cancer patients with advanced disease will develop bone metastases. Once established in bone, tumor cells produce factors that cause changes in normal bone remodeling, such as parathyroid hormone-related protein (PTHrP). While enhanced expression of PTHrP is known to stimulate osteoclasts to resorb bone, the environmental factors driving tumor cells to express PTHrP in the early stages of development of metastatic bone disease are unknown. In this study, we have shown that tumor cells known to metastasize to bone respond to 2D substrates with rigidities comparable to that of the bone microenvironment by increasing expression and production of PTHrP. The cellular response is regulated by Rho-dependent actomyosin contractility mediated by TGF-ß signaling. Inhibition of Rho-associated kinase (ROCK) using both pharmacological and genetic approaches decreased PTHrP expression. Furthermore, cells expressing a dominant negative form of the TGF-ß receptor did not respond to substrate rigidity, and inhibition of ROCK decreased PTHrP expression induced by exogenous TGF-ß. These observations suggest a role for the differential rigidity of the mineralized bone microenvironment in early stages of tumor-induced osteolysis, which is especially important in metastatic cancer since many cancers (such as those of the breast and lung) preferentially metastasize to bone. PMID:21085597

  11. Aldosterone and myocardial extracellular matrix expansion in type 2 diabetes mellitus.

    PubMed

    Rao, Ajay D; Shah, Ravi V; Garg, Rajesh; Abbasi, Siddique A; Neilan, Tomas G; Perlstein, Todd S; Di Carli, Marcelo F; Jerosch-Herold, Michael; Kwong, Raymond Y; Adler, Gail K

    2013-07-01

    Myocardial extracellular matrix expansion and reduced coronary flow reserve (CFR) occur in patients with type 2 diabetes mellitus without heart failure or coronary artery disease. Because aldosterone is implicated in the pathophysiology of cardiac fibrosis and vascular injury, the aim of this study was to test the hypothesis that aldosterone is associated with extracellular matrix expansion and reduced CFR in type 2 diabetes mellitus. Patients with type 2 diabetes mellitus without evidence of coronary artery disease were recruited. Blood pressure, lipid management, and glycemic control were optimized over 3 months. Cardiac magnetic resonance imaging with T1 mapping was used to measure myocardial extracellular volume (ECV). Cardiac positron emission tomography was used to assess CFR. On a liberal, 250 mEq/day sodium diet, 24-hour urinary aldosterone and change in serum aldosterone with angiotensin II stimulation were measured. Fifty-three participants with type 2 diabetes (68% men, mean age 53 ± 7 years, mean body mass index 32.2 ± 4.3 kg/m², mean glycosylated hemoglobin 6.8 ± 0.7%, mean systolic blood pressure 126 ± 14 mm Hg) without infarction or ischemia by cardiac magnetic resonance and positron emission tomography were studied. Subjects had impaired CFR (2.51 ± 0.83) and elevated ECV (0.36 ± 0.05), despite normal echocardiographic diastolic function and normal left ventricular function. Myocardial ECV, but not CFR, was positively associated with 24-hour urinary aldosterone excretion (r = 0.37, p = 0.01) and angiotensin II-stimulated aldosterone increase (r = 0.35, p = 0.02). In a best-overall multivariate model (including age, gender, body mass index, glycosylated hemoglobin, and blood pressure), 24-hour urinary aldosterone was the strongest predictor of myocardial ECV (p = 0.004). In conclusion, in patients with type 2 diabetes mellitus without coronary artery disease, aldosterone is associated with myocardial extracellular matrix expansion. These

  12. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix.

    PubMed

    Borlee, Bradley R; Goldman, Aaron D; Murakami, Keiji; Samudrala, Ram; Wozniak, Daniel J; Parsek, Matthew R

    2010-02-01

    Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a beta-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural

  13. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix

    PubMed Central

    Borlee, Bradley R; Goldman, Aaron D; Murakami, Keiji; Samudrala, Ram; Wozniak, Daniel J; Parsek, Matthew R

    2010-01-01

    Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a β-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural

  14. Right ventricular function after repair of tetralogy of Fallot: a comparison between bovine pericardium and porcine small intestinal extracellular matrix.

    PubMed

    Naik, Ronak; Johnson, Jason; Kumar, T K S; Philip, Ranjit; Boston, Umar; Knott-Craig, Christopher J

    2017-05-29

    The porcine small intestinal extracellular matrix reportedly has the potential to differentiate into viable myocardial cells. When used in tetralogy of Fallot repair, it may improve right ventricular function. We evaluated right ventricular function after repair of tetralogy of Fallot with extracellular matrix versus bovine pericardium. Subjects with non-transannular repair of tetralogy of Fallot with at least 1 year of follow-up were selected. The extracellular matrix and bovine pericardium groups were compared. We used three-dimensional right ventricular ejection fraction, right ventricle global longitudinal strain, and tricuspid annular plane systolic excursion to assess right ventricular function. The extracellular matrix group had 11 patients, whereas the bovine pericardium group had 10 patients. No differences between the groups were found regarding sex ratio, age at surgery, and cardiopulmonary bypass time. The follow-up period was 28±12.6 months in the extracellular matrix group and 50.05±17.6 months in the bovine pericardium group (p=0.001). The mean three-dimensional right ventricular ejection fraction (55.7±5.0% versus 55.3±5.2%, p=0.73), right ventricular global longitudinal strain (-18.5±3.0% versus -18.0±2.2%, p=0.44), and tricuspid annular plane systolic excursions (1.59±0.16 versus 1.59±0.2, p=0.93) were similar in the extracellular matrix group and in the bovine pericardium group, respectively. Right ventricular global longitudinal strain in healthy children is reported at -29±3% in literature. In a small cohort of the patients undergoing non-transannular repair of tetralogy of Fallot, there was no significant difference in right ventricular function between groups having extracellular matrix versus bovine pericardium patches followed-up for more than 1 year. Lower right ventricular longitudinal strain noted in both the groups compared to healthy children.

  15. The extracellular matrix is a novel attribute of endothelial progenitors and of hypoxic mature endothelial cells

    PubMed Central

    Kusuma, Sravanti; Zhao, Stephen; Gerecht, Sharon

    2012-01-01

    Extracellular matrix (ECM) production is critical to preserve the function and integrity of mature blood vessels. Toward the engineering of blood vessels, studies have centered on ECM production by supporting cells, whereas few studies implicate endothelial cells (ECs) with ECM synthesis. Here, we elucidate variations between cultured human arterial, venous, and progenitor ECs with respect to ECM deposition assembly, composition, and response to biomolecular and physiological factors. Our studies reveal that progenitor ECs, endothelial colony-forming cells (ECFCs), deposit collagen IV, fibronectin, and laminin that assemble to an organized weblike structure, as confirmed by decellularized cultures. Mature ECs only express these ECM proteins intracellularly. ECFC-derived ECM is abrogated in response to TGFβ signaling inhibition and actin cytoskeleton disruption. Hypoxic (1%) and physiological (5%) O2 tension stimulate ECM deposition from mature ECs. Interestingly, deposition of collagen I is observed only under 5% O2 tension. ECM production from all ECs is found to be regulated by hypoxia-inducible factors 1α and 2α but differentially in the different cell lines. Collectively, we suggest that ECM deposition and assembly by ECs is dependent on maturation stage and oxygen supply and that these findings can be harnessed to advance engineered vascular therapeutics.—Kusuma, S., Zhao, S., Gerecht, S. The extracellular matrix is a novel attribute of endothelial progenitors and of hypoxic mature endothelial cells. PMID:22919069

  16. Does extracellular matrix of the varicose vein wall change according to clinical stage?

    PubMed

    Demirkıran, Mehmet Ali; Köksoy, Cüneyt; Heper, Aylin Okçu; Bengisun, Uğur

    2014-01-01

    The etiology and pathophysiology of chronic venous disease is not fully understood. This study aimed to determine the variation of the extracellular matrix proteins in varicose vein wall according to clinical stage. Forty varicose and 10 control veins were sampled from the saphenofemoral junction. The Clinical Etiologic Anatomic Pathophysiologic (CEAP) classification was used in patients with varicose veins. Samples were stained with hematoxylin-eosin, Masson's trichrome, EVG (Elastica-van Gieson) stain and with laminin, fibronectin, tenascin antibodies. Stained samples were examined immuno-histochemically. Changes in extracellular matrix were determined semi-quantitatively using light microscopy. It was observed that in the early stages (C2-C3) of chronic venous disease, fibrosis is increased in the intima and media layers, with fragmentation in lamina elastica interna, and increased tenascin expression in the intima layer. In advanced stages (C4-C6), the accumulation of tenascin in the intima continued along with fibrosis in the media layer, the thickness of the media layer increased and fibronectin deposition was observed. This study showed that changes first occur in the intima during the early stages of the disease with addition of alterations in the media layer at later stages.

  17. Does extracellular matrix of the varicose vein wall change according to clinical stage?

    PubMed Central

    Demirkıran, Mehmet Ali; Köksoy, Cüneyt; Heper, Aylin Okçu; Bengisun, Uğur

    2014-01-01

    Objective: The etiology and pathophysiology of chronic venous disease is not fully understood. This study aimed to determine the variation of the extracellular matrix proteins in varicose vein wall according to clinical stage. Material and Methods: Forty varicose and 10 control veins were sampled from the saphenofemoral junction. The Clinical Etiologic Anatomic Pathophysiologic (CEAP) classification was used in patients with varicose veins. Samples were stained with hematoxylin-eosin, Masson’s trichrome, EVG (Elastica-van Gieson) stain and with laminin, fibronectin, tenascin antibodies. Stained samples were examined immuno-histochemically. Changes in extracellular matrix were determined semi-quantitatively using light microscopy. Results: It was observed that in the early stages (C2–C3) of chronic venous disease, fibrosis is increased in the intima and media layers, with fragmentation in lamina elastica interna, and increased tenascin expression in the intima layer. In advanced stages (C4–C6), the accumulation of tenascin in the intima continued along with fibrosis in the media layer, the thickness of the media layer increased and fibronectin deposition was observed. Conclusion: This study showed that changes first occur in the intima during the early stages of the disease with addition of alterations in the media layer at later stages. PMID:25931926

  18. Up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver

    PubMed Central

    Zhang, Shuai; Li, Tao-Sheng; Soyama, Akihiko; Tanaka, Takayuki; Yan, Chen; Sakai, Yusuke; Hidaka, Masaaki; Kinoshita, Ayaka; Natsuda, Koji; Fujii, Mio; Kugiyama, Tota; Baimakhanov, Zhassulan; Kuroki, Tamotsu; Gu, Weili; Eguchi, Susumu

    2016-01-01

    Although the healthy liver is known to have high regenerative potential, poor liver regeneration under pathological conditions remains a substantial problem. We investigated the key molecules that impair the regeneration of cholestatic liver. C57BL/6 mice were randomly subjected to partial hepatectomy and bile duct ligation (PH+BDL group, n = 16), partial hepatectomy only (PH group, n = 16), or sham operation (Sham group, n = 16). The liver sizes and histological findings were similar in the PH and sham groups 14 days after operation. However, compared with those in the sham group, the livers in mice in the PH+BDL group had a smaller size, a lower cell proliferative activity, and more fibrotic tissue 14 days after the operation, suggesting the insufficient regeneration of the cholestatic liver. Pathway-focused array analysis showed that many genes were up- or down-regulated over 1.5-fold in both PH+BDL and PH groups at 1, 3, 7, and 14 days after treatment. Interestingly, more genes that were functionally related to the extracellular matrix and inflammatory chemokines were found in the PH+BDL group than in the PH group at 7 and 14 days after treatment. Our data suggest that up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver. PMID:27226149

  19. Bubaline Cholecyst Derived Extracellular Matrix for Reconstruction of Full Thickness Skin Wounds in Rats

    PubMed Central

    Shakya, Poonam; Sharma, A. K.; Kumar, Naveen; Vellachi, Remya; Mathew, Dayamon D.; Dubey, Prasoon; Singh, Kiranjeet; Shrivastava, Sonal; Shrivastava, Sameer; Maiti, S. K.; Hasan, Anwarul; Singh, K. P.

    2016-01-01

    An acellular cholecyst derived extracellular matrix (b-CEM) of bubaline origin was prepared using anionic biological detergent. Healing potential of b-CEM was compared with commercially available collagen sheet (b-CS) and open wound (C) in full thickness skin wounds in rats. Thirty-six clinically healthy adult Sprague Dawley rats of either sex were randomly divided into three equal groups. Under general anesthesia, a full thickness skin wound (20 × 20 mm2) was created on the dorsum of each rat. The defect in group I was kept as open wound and was taken as control. In group II, the defect was repaired with commercially available collagen sheet (b-CS). In group III, the defect was repaired with cholecyst derived extracellular matrix of bovine origin (b-CEM). Planimetry, wound contracture, and immunological and histological observations were carried out to evaluate healing process. Significantly (P < 0.05) increased wound contraction was observed in b-CEM (III) as compared to control (I) and b-CS (II) on day 21. Histologically, improved epithelization, neovascularization, fibroplasia, and best arranged collagen fibers were observed in b-CEM (III) as early as on postimplantation day 21. These findings indicate that b-CEM have potential for biomedical applications for full thickness skin wound repair in rats. PMID:27127678

  20. Trafficking mechanisms of extracellular matrix macromolecules: insights from vertebrate development and human diseases.

    PubMed

    Unlu, Gokhan; Levic, Daniel S; Melville, David B; Knapik, Ela W

    2014-02-01

    Cellular life depends on protein transport and membrane traffic. In multicellular organisms, membrane traffic is required for extracellular matrix deposition, cell adhesion, growth factor release, and receptor signaling, which are collectively required to integrate the development and physiology of tissues and organs. Understanding the regulatory mechanisms that govern cargo and membrane flow presents a prime challenge in cell biology. Extracellular matrix (ECM) secretion remains poorly understood, although given its essential roles in the regulation of cell migration, differentiation, and survival, ECM secretion mechanisms are likely to be tightly controlled. Recent studies in vertebrate model systems, from fishes to mammals and in human patients, have revealed complex and diverse loss-of-function phenotypes associated with mutations in components of the secretory machinery. A broad spectrum of diseases from skeletal and cardiovascular to neurological deficits have been linked to ECM trafficking. These discoveries have directly challenged the prevailing view of secretion as an essential but monolithic process. Here, we will discuss the latest findings on mechanisms of ECM trafficking in vertebrates. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Diffusion of Particles in the Extracellular Matrix: The Effect of Repulsive Electrostatic Interactions

    PubMed Central

    Stylianopoulos, Triantafyllos; Poh, Ming-Zher; Insin, Numpon; Bawendi, Moungi G.; Fukumura, Dai; Munn, Lance L.; Jain, Rakesh K.

    2010-01-01

    Diffusive transport of macromolecules and nanoparticles in charged fibrous media is of interest in many biological applications, including drug delivery and separation processes. Experimental findings have shown that diffusion can be significantly hindered by electrostatic interactions between the diffusing particle and charged components of the extracellular matrix. The implications, however, have not been analyzed rigorously. Here, we present a mathematical framework to study the effect of charge on the diffusive transport of macromolecules and nanoparticles in the extracellular matrix of biological tissues. The model takes into account steric, hydrodynamic, and electrostatic interactions. We show that when the fiber size is comparable to the Debye length, electrostatic forces between the fibers and the particles result in slowed diffusion. However, as the fiber diameter increases the repulsive forces become less important. Our results explain the experimental observations that neutral particles diffuse faster than charged particles. Taken together, we conclude that optimal particles for delivery to tumors should be initially cationic to target the tumor vessels and then change to neutral charge after exiting the blood vessels. PMID:20816045

  2. Evidence of Innervation following Extracellular Matrix Scaffold Mediated Remodeling of Muscular Tissues

    PubMed Central

    Agrawal, Vineet; Brown, Bryan N.; Beattie, Allison J.; Gilbert, Thomas W.; Badylak, Stephen F.

    2009-01-01

    Naturally occurring porcine derived extracellular matrix (ECM) has successfully been used as a biologic scaffold material for site-specific reconstruction of a wide variety of tissues. The site-specific remodeling process includes rapid degradation of the scaffold with concomitant recruitment of mononuclear cells, endothelial cells, and bone marrow derived cells, and can lead to formation of functional skeletal and smooth muscle tissue. However, the temporal and spatial patterns of innervation of the remodeling scaffold material in muscular tissues are not well understood. A retrospective study was conducted to investigate the presence of nervous tissue in a rat model of abdominal wall reconstruction and a canine model of esophageal reconstruction in which ECM scaffolds were used as inductive scaffolds. Evidence of mature nerve, immature nerve, and Schwann cells was found within the remodeled ECM at 28 days in the rat body wall model, and at 91 days post surgery in a canine model of esophageal repair. Additionally, a microscopic and morphologic study that investigated the response of primary cultured neurons seeded upon an ECM scaffold showed that neuronal survival and outgrowth was supported by the ECM substrate. Finally, matricryptic peptides resulting from rapid degradation of the ECM scaffold induced migration of terminal Schwann cells in a concentration dependent fashion in vitro. The findings of this study suggest that the reconstruction of tissues in which innervation is an important functional component is possible with use of biologic scaffolds composed of extracellular matrix. PMID:19701935

  3. Effects of Extracellular Matrix Density and Mesenchymal Stem Cells on Neovascularization In Vivo

    PubMed Central

    Kniazeva, Ekaterina; Kachgal, Suraj

    2011-01-01

    Aberrant angiogenesis is common to a variety of diseases in which alterations in tissue mechanical properties also occur. A fundamental understanding of the interdependence of angiogenesis and tissue structural properties may enhance the development of therapeutic strategies. We previously established that increasing extracellular matrix density inhibits capillary morphogenesis in three-dimensional tissues in vitro, and that addition of human mesenchymal stem cells (MSCs) partially rescues a healthy angiogenic phenotype. This study's goal was to investigate if these effects can be recapitulated in vivo. Human umbilical vein endothelial cells, MSCs, or a mixture of both was suspended in fibrin gel precursor solutions of 5, 10, and 15 mg/mL concentrations and injected subcutaneously into SCID mice. Neovascularization was assessed in tissue constructs retrieved at 3, 7, and 21 days by quantifying vessel numbers, perfusion, thickness, maturity, and perivascular collagen deposition. The data show that changing extracellular matrix density inhibits capillary morphogenesis in vivo in a manner consistent with that observed in vitro. Delivery of both human umbilical vein endothelial cells and MSCs produced more robust and mature vessels than delivery of either cell type alone in all tissue concentrations. PMID:20979533

  4. Sesamin inhibits lipopolysaccharide-induced inflammation and extracellular matrix catabolism in rat intervertebral disc.

    PubMed

    Li, Kang; Li, Yan; Xu, Bo; Mao, Lu; Zhao, Jie

    2016-09-01

    Intervertebral disc (IVD) degeneration contributes to most spinal degenerative diseases, while treatment inhibiting IVD degeneration is still in the experimental stage. Sesamin, a bioactive component extracted from sesame, has been reported to exert chondroprotective and anti-inflammatory effects. Here, we analyzed the anti-inflammatory and anti-catabolic effects of sesamin on rat IVD in vitro and ex vivo. Results show that sesamin significantly inhibits the lipopolysaccharide (LPS)-induced expression of catabolic enzymes (MMP-1, MMP-3, MMP-13, ADAMTS-4, ADAMTS-5) and inflammation factors (IL-1β, TNF-α, iNOS, NO, COX-2, PGE2) in a dose-dependent manner in vitro. It is also proven that migration of macrophages induced by LPS can be inhibited by treatment with sesamin. Organ culture experiments demonstrate that sesamin protects the IVD from LPS-induced depletion of the extracellular matrix ex vivo. Moreover, sesamin suppresses LPS-induced activation of the mitogen-activated protein kinase (MAPK) pathway through inhibiting phosphorylation of JNK, the common downstream signaling pathway of LPS and IL-1β, which may be the potential mechanism of the effects of sesamin. In light of our results, sesamin protects the IVD from inflammation and extracellular matrix catabolism, presenting positive prospects in the treatment of IVD degenerative diseases.

  5. Ultrastructure of the extracellular matrix of bovine dura mater, optic nerve sheath and sclera.

    PubMed Central

    Raspanti, M; Marchini, M; Della Pasqua, V; Strocchi, R; Ruggeri, A

    1992-01-01

    The sclera, the outermost sheath of the optic nerve and the dura mater have been investigated histologically and ultrastructurally. Although these tissues appear very similar under the light microscope, being dense connective tissues mainly composed of collagen bundles and a limited amount of cells and elastic fibres, they exhibit subtle differences on electron microscopy. In the dura and sclera collagen appears in the form of large, nonuniform fibrils, similar to those commonly found in tendons, while in the optic nerve sheath the fibrils appear smaller and uniform, similar to those commonly observed in reticular tissues, vessel walls and skin. Freeze-fracture also reveals these fibrils to have different subfibrillar architectures, straight or helical, which correspond to 2 distinct forms of collagen fibril previously described (Raspanti et al. 1989). The other extracellular matrix components also vary with the particular collagen fibril structure. Despite their common embryological derivation, the dura mater, optic nerve sheath and sclera exhibit diversification of their extracellular matrix consistent with the mechanical loads to which these tissues are subjected. Our observations indicate that the outermost sheath of the optic nerve resembles the epineurium of peripheral nerves rather than the dura to which it is commonly likened. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:1295858

  6. Putative chitin synthases from Branchiostoma floridae show extracellular matrix-related domains and mosaic structures.

    PubMed

    Guerriero, Gea

    2012-08-01

    The transition from unicellular to multicellular life forms requires the development of a specialized structural component, the extracellular matrix (ECM). In Metazoans, there are two main supportive systems, which are based on chitin and collagen/hyaluronan, respectively. Chitin is the major constituent of fungal cell walls and arthropod exoskeleton. However, presence of chitin/chitooligosaccharides has been reported in lower chordates and during specific stages of vertebrate development. In this study, the occurrence of chitin synthases (CHSs) was investigated with a bioinformatics approach in the cephalochordate Branchiostoma floridae, in which the presence of chitin was initially reported in the skeletal rods of the pharyngeal gill basket. Twelve genes coding for proteins containing conserved amino acid residues of processive glycosyltransferases from GT2 family were found and 10 of them display mosaic structures with novel domains never reported previously in a chitin synthase. In particular, the presence of a discoidin (DS) and a sterile alpha motif (SAM) domain was found in nine identified proteins. Sequence analyses and homology modelling suggest that these domains might interact with the extracellular matrix and mediate protein-protein interaction. The multi-domain putative chitin synthases from B. floridae constitute an emblematic example of the explosion of domain innovation and shuffling which predate Metazoans.

  7. Mesenchymal Remodeling during Palatal Shelf Elevation Revealed by Extracellular Matrix and F-Actin Expression Patterns

    PubMed Central

    Chiquet, Matthias; Blumer, Susan; Angelini, Manuela; Mitsiadis, Thimios A.; Katsaros, Christos

    2016-01-01

    During formation of the secondary palate in mammalian embryos, two vertically oriented palatal shelves rapidly elevate into a horizontal position above the tongue, meet at the midline, and fuse to form a single entity. Previous observations suggested that elevation occurs by a simple 90° rotation of the palatal shelves. More recent findings showed that the presumptive midline epithelial cells are not located at the tips of palatal shelves before elevation, but mostly toward their medial/lingual part. This implied extensive tissue remodeling during shelf elevation. Nevertheless, it is still not known how the shelf mesenchyme reorganizes during this process, and what mechanism drives it. To address this question, we mapped the distinct and restricted expression domains of certain extracellular matrix components within the developing palatal shelves. This procedure allowed to monitor movements of entire mesenchymal regions relative to each other during shelf elevation. Consistent with previous notions, our results confirm a flipping movement of the palatal shelves anteriorly, whereas extensive mesenchymal reorganization is observed more posteriorly. There, the entire lingual portion of the vertical shelves moves close to the midline after elevation, whereas the mesenchyme at the original tip of the shelves ends up ventrolaterally. Moreover, we observed that the mesenchymal cells of elevating palatal shelves substantially align their actin cytoskeleton, their extracellular matrix, and their nuclei in a ventral to medial direction. This indicates that, like in other morphogenetic processes, actin-dependent cell contractility is a major driving force for mesenchymal tissue remodeling during palatogenesis. PMID:27656150

  8. Trafficking Mechanisms of Extracellular Matrix Macromolecules: Insights from Vertebrate Development and Human Diseases

    PubMed Central

    Unlu, Gokhan; Levic, Daniel S.; Melville, David B.; Knapik, Ela W.

    2014-01-01

    Cellular life depends on protein transport and membrane traffic. In multicellular organisms, membrane traffic is required for extracellular matrix deposition, cell adhesion, growth factor release, and receptor signaling, which are collectively required to integrate the development and physiology of tissues and organs. Understanding the regulatory mechanisms that govern cargo and membrane flow presents a prime challenge in cell biology. Extracellular matrix (ECM) secretion remains poorly understood, although given its essential roles in the regulation of cell migration, differentiation, and survival, ECM secretion mechanisms are likely to be tightly controlled. Recent studies in vertebrate model systems, from fishes to mammals and in human patients, have revealed complex and diverse loss-of-function phenotypes associated with mutations in components of the secretory machinery. A broad spectrum of diseases from skeletal and cardiovascular to neurological deficits have been linked to ECM trafficking. These discoveries have directly challenged the prevailing view of secretion as an essential but monolithic process. Here, we will discuss the latest findings on mechanisms of ECM trafficking in vertebrates. PMID:24333299

  9. Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms

    PubMed Central

    Martins, Margarida; Uppuluri, Priya; Thomas, Derek P.; Cleary, Ian A.; Henriques, Mariana; Lopez-Ribot, José L.; Oliveira, Rosário

    2014-01-01

    DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C.albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance. PMID:20012895

  10. Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

    PubMed

    Shimada, Nao; Nishio, Keiko; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

    2004-10-01

    Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins.

  11. Transitional Remodeling of the Hepatic Extracellular Matrix in Alcohol-Induced Liver Injury

    PubMed Central

    Poole, Lauren G.

    2016-01-01

    Alcohol consumption is a common custom worldwide, and the toxic effects of alcohol on several target organs are well understood. The liver is the primary site of alcohol metabolism and is therefore the major target of alcohol toxicity. Alcoholic liver disease is a spectrum of disease states, ranging from simple steatosis (fat accumulation), to inflammation, and eventually to fibrosis and cirrhosis if untreated. The fibrotic stage of ALD is primarily characterized by robust accumulation of extracellular matrix (ECM) proteins (collagens) which ultimately impairs the function of the organ. The role of the ECM in early stages of ALD is poorly understood, but recent research has demonstrated that a number of changes in the hepatic ECM in prefibrotic ALD not only are present, but may also contribute to disease progression. The purpose of this review is to summarize the established and proposed changes to the hepatic extracellular matrix (ECM) that may contribute to earlier stages of ALD development and to discuss potential mechanisms by which these changes may mediate the progression of the disease. PMID:27843941

  12. Skeletal muscle extracellular matrix remodelling after aestivation in the green striped burrowing frog, Cyclorana alboguttata.

    PubMed

    Hudson, Nicholas J; Harper, Gregory S; Allingham, Peter G; Franklin, Craig E; Barris, W; Lehnert, Sigrid A

    2007-03-01

    Connective tissue has recently been found to play a role in mediating mammalian skeletal muscle atrophy. We investigated connective tissue remodelling in the skeletal muscle of a species of the Australian burrowing frog, Cyclorana alboguttata. Despite being inactive whilst aestivating, the frog shows an inhibition of muscle atrophy. Connective tissue size and distribution was measured in histological sections of the cruralis muscle of control and aestivating C. alboguttata. Using a custom written software application we could detect no significant difference in any connective tissue morphological parameter between the two treatment groups. Biochemical measurements of gelatinase activity showed 2-fold higher activity in aestivating gastrocnemius muscle than in controls (p<0.001). We measured the messenger RNA transcript levels for C. alboguttata metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase 2 (TIMP2) in cruralis skeletal muscle using quantitative real-time PCR. The trend of reduced expression of the two genes in the aestivators did not meet statistical significance. This work indicates that aestivation in C. alboguttata leads to subtle and specific changes in some extracellular matrix remodelling factors. Their main impact is to maintain proportional representation of extracellular matrix components of skeletal muscle and therefore preserve the active frog phenotype.

  13. Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation

    PubMed Central

    Yu, L.; Grist, S. M.; Nasseri, S. S.; Ni, C.; Cheung, K. C.

    2015-01-01

    Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture. PMID:25945144

  14. UV irradiation and desiccation modulate the three-dimensional extracellular matrix of Nostoc commune (Cyanobacteria).

    PubMed

    Wright, Deborah J; Smith, Sue C; Joardar, Vinita; Scherer, Siegfried; Jervis, Jody; Warren, Andrew; Helm, Richard F; Potts, Malcolm

    2005-12-02

    Cyanobacterium Nostoc commune can tolerate the simultaneous stresses of desiccation, UV irradiation, and oxidation. Acidic WspA, of approximately 33.6 kDa, is secreted to the three-dimensional extracellular matrix and accounts for greater than 70% of the total soluble protein. The wspA gene of N. commune strain DRH1 was cloned and found in a single genomic copy, in a monocistronic operon. Transcription of wspA and sodF (superoxide dismutase), and synthesis and secretion of WspA, were induced upon desiccation or UV-A/B irradiation of cells. Recombinant WspA binds the UV-A/B absorbing pigment scytonemin through non-covalent interactions. WspA peptide polymorphism, and heterogeneity of multiple wspA sequences within cells of a single colony, account for distinct WspA isoforms. WspA has no similarity to entries in the sequence databases and wspA, a possible xenolog, is restricted to a subset of strains in the "form species" N. commune characterized through group I intron phylogeny. We hypothesize that WspA plays a central role in the global stress response of N. commune through modulation of the structure and function of the three-dimensional extracellular matrix, particularly the transport, distribution, and/or macromolecular architecture of mycosporine and scytonemin UV-A/B absorbing pigment complexes.

  15. Extracellular matrix remodelling in myocardial hypertrophy and failure : focus on osteopontin.

    PubMed

    Francia, Pietro; Uccellini, Arianna; Frattari, Alessandra; Modestino, Anna; Ricotta, Agnese; Balla, Cristina; Scialla, Ludovica; Volpe, Massimo

    2009-12-01

    Cardiac remodelling refers to molecular and cellular changes of the myocardium, as well as adapting alterations in size, shape and function of the heart in response to changing loading conditions. It represents the final common pathway of different heart diseases, and is recognized as a crucial aspect of cardiac and myocardial dysfunction and a well established determinant of the clinical course of heart failure.Osteopontin is an extracellular matrix glycoprotein secreted by osteoblasts, osteoclasts, macrophages, T cells, vascular smooth muscle cells, fibroblasts and cardiomyocytes. Osteopontin is not expressed in healthy cardiac tissue, although its expression can be triggered by pressure or volume overload, hypoxia and angiotensin II. Indeed, osteopontin has been reported in macrophages and interstitial tissues early after myocardial infarction and in cardiac macrophage-like cells of inflammatory lesions in experimental models of cardiomyopathy. Pressure overload is associated with osteopontin overexpression as well. Indeed, myocardial osteopontin messenger RNA is upregulated in rats following renovascular hypertension or aortic banding. In humans, a significant correlation exists between increased osteopontin immunoreactivity in cardiac myocytes and impaired left ventricular function or cardiomyocyte hypertrophy in patients with dilated cardiomyopathy.The present article focuses on the role of osteopontin in myocardial hypertrophy and remodelling. In general, evidence supports the concept that osteopontin plays a crucial role in extracellular matrix remodelling following myocardial adaptation to hypertrophic, inflammatory and neurohormonal stimuli in the overloaded heart.

  16. Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms.

    PubMed

    Martins, Margarida; Uppuluri, Priya; Thomas, Derek P; Cleary, Ian A; Henriques, Mariana; Lopez-Ribot, José L; Oliveira, Rosário

    2010-05-01

    DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.

  17. A Novel Method for Simulating the Extracellular Matrix in Models of Tumour Growth

    PubMed Central

    Toma, Alina; Mang, Andreas; Schuetz, Tina A.; Becker, Stefan; Buzug, Thorsten M.

    2012-01-01

    A novel hybrid continuum-discrete model to simulate tumour growth on a cellular scale is proposed. The lattice-based spatiotemporal model consists of reaction-diffusion equations that describe interactions between cancer cells and their microenvironment. The fundamental ingredients that are typically considered are the nutrient concentration, the extracellular matrix (ECM), and matrix degrading enzymes (MDEs). The in vivo processes are very complex and occur on different levels. This in turn leads to huge computational costs. The main contribution of the present work is therefore to describe the processes on the basis of simplified mathematical approaches, which, at the same time, depict realistic results to understand the biological processes. In this work, we discuss if we have to simulate the MDE or if the degraded matrix can be estimated directly with respect to the cancer cell distribution. Additionally, we compare the results for modelling tumour growth using the common and our simplified approach, thereby demonstrating the advantages of the proposed method. Therefore, we introduce variations of the positioning of the nutrient delivering blood vessels and use different initializations of the ECM. We conclude that the novel method, which does not explicitly model the matrix degrading enzymes, provides means for a straightforward and fast implementation for modelling tumour growth. PMID:22919426

  18. Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment.

    PubMed

    Palchesko, Rachelle N; Szymanski, John M; Sahu, Amrita; Feinberg, Adam W

    2014-09-01

    Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments.

  19. Effects of extracellular fiber architecture on cell membrane shear stress in a 3D fibrous matrix.

    PubMed

    Pedersen, John A; Boschetti, Federica; Swartz, Melody A

    2007-01-01

    Interstitial fluid flow has been shown to affect the organization and behavior of cells in 3D environments in vivo and in vitro, yet the forces driving such responses are not clear. Due to the complex architecture of the extracellular matrix (ECM) and the difficulty of measuring fluid flow near cells embedded in it, the levels of shear stress experienced by cells in this environment are typically estimated using bulk-averaged matrix parameters such as hydraulic permeability. While this is useful for estimating average stresses, it cannot yield insight into how local matrix fiber architecture-which is cell-controlled in the immediate pericellular environment-affects the local stresses imposed on the cell surface. To address this, we used computational fluid dynamics to study flow through an idealized mesh constructed of a cubic lattice of fibers simulating a typical in vitro collagen gel. We found that, in such high porosity matrices, the fibers strongly affect the flow fields near the cell, with peak shear stresses up to five times higher than those predicted by the Brinkman equation. We also found that minor remodeling of the fibers near the cell surface had major effects on the shear stress profile on the cell. These findings demonstrate the importance of fiber architecture to the fluid forces on a cell embedded in a 3D matrix, and also show how small modifications in the local ECM can lead to large changes in the mechanical environment of the cell.

  20. Dendritic cell podosomes are protrusive and invade the extracellular matrix using metalloproteinase MMP-14

    PubMed Central

    Gawden-Bone, Christian; Zhou, Zhongjun; King, Emma; Prescott, Alan; Watts, Colin; Lucocq, John

    2010-01-01

    Podosomes are spot-like actin-rich structures formed at the ventral surface of monocytic and haematopoietic cells. Podosomes degrade extracellular matrix and are proposed to be involved in cell migration. A key question is whether podosomes form protrusions similar to the invadopodia of cancer cells. We characterised podosomes of immature dendritic cells using electron microscopy combined with both conventional and novel high-resolution structured illumination light microscopy. Dendritic cell podosomes are composed of actin foci surrounded by a specialised ring region that is rich in material containing paxillin. We found that podosomes were preferential sites for protrusion into polycarbonate filters impregnated with crosslinked gelatin, degrading up to 2 μm of matrix in 24 hours. Podosome-associated uptake of colloidal gold-labelled gelatin matrix appeared to occur via large phagosome-like structures or narrow tubular invaginations. The motor protein myosin-II was excluded from ring or core regions but was concentrated around them and the myosin-II inhibitor Blebbistatin reduced the length of podosome protrusions. Finally, we found that degradation, protrusion and endocytosis in this system are dependent on the matrix metalloproteinase MMP-14. We propose that podosomes mediate migration of dendritic cells through tissues by means of myosin-II-dependent protrusion coupled to MMP-14-dependent degradation and endocytosis. PMID:20356925

  1. The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Billings, Nicole; Ramirez Millan, Maria; Caldara, Marina; Rusconi, Roberto; Tarasova, Yekaterina; Stocker, Roman; Ribbeck, Katharina

    2013-01-01

    Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development. PMID:23950711

  2. Ubiquitylation functions in the calcium carbonate biomineralization in the extracellular matrix.

    PubMed

    Fang, Dong; Pan, Cong; Lin, Huijuan; Lin, Ya; Xu, Guangrui; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2012-01-01

    Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes.

  3. Ubiquitylation Functions in the Calcium Carbonate Biomineralization in the Extracellular Matrix

    PubMed Central

    Fang, Dong; Pan, Cong; Lin, Huijuan; Lin, Ya; Xu, Guangrui; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2012-01-01

    Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes. PMID:22558208

  4. Breast tumour initiating cell fate is regulated by microenvironmental cues from an extracellular matrix.

    PubMed

    Saha, Sharmistha; Lo, Pang-Kuo; Duan, Xinrui; Chen, Hexin; Wang, Qian

    2012-08-01

    Cancer stem cells, also known as tumour-initiating cells (TICs), are identified as highly tumorigenic population within tumours and hypothesized to be main regulators in tumour growth, metastasis and relapse. Evidence also suggests that a tumour microenvironment plays a critical role in the development and progression of cancer, by constantly modulating cell-matrix interactions. Scientists have tried to characterize and identify the TIC population but the actual combination of extracellular components in deciphering the fate of TICs has not been explored. The basic unanswered question is the phenotypic stability of this TIC population in a tissue extracellular matrix setting. The in vivo complexity makes it difficult to identify parameters in a diverse milieu that affect TICs behaviour. Herein we studied how the TIC population would respond when subjected to a unique microenvironment composed of different extracellular proteins. The TIC-enriched population isolated from a Her2/neu-induced mouse mammary tumour was cultured on collagen, fibronectin and laminin coated substrates for one to two weeks. Our observations indicate that a laminin substrate can maintain the majority of the self-renewing and tumorigenic TIC population, whereas collagen induced a more differentiated phenotype of the cells. Also interestingly, fibronectin substrates dictated an invasive phenotype of TICs as evidenced from the EMT-related gene expression pattern. The results of this study signify that the microenvironmental cues play a considerable role in tumour relapse and progression by altering the cancer stem cell behaviour and thus this knowledge could be used to design novel cancer therapeutics.

  5. Extracellular citrullination inhibits the function of matrix associated TGF-β.

    PubMed

    Sipilä, Kalle H; Ranga, Vipin; Rappu, Pekka; Torittu, Annamari; Pirilä, Laura; Käpylä, Jarmo; Johnson, Mark S; Larjava, Hannu; Heino, Jyrki

    2016-09-01

    In inflammatory arthritis peptidyl arginine deiminase (PAD) enzymes can citrullinate arginine residues in extracellular matrix (ECM) proteins, such as collagens and fibronectin. This may lead to the generation of anti-citrullinated protein antibodies, important diagnostic markers in rheumatoid arthritis. In addition, the citrullination may directly affect protein function. Based on structural analysis, we found that most ECM-associated growth factors (GFs) have arginine residues in their receptor recognition sites. Thus, they are potential functional targets of extracellular citrullination. To examine this further, we focused on the citrullination of transforming growth factor-βs (TGF-β), well-known ECM-associated GFs. PAD-treatment of CHO-LTBP1 cell derived matrix, rich with TGF-β, decreased the level of TGF-β activity as detected by HaCaT and MLEC-PAI-1/Lu reporter cells. Additional experiments indicated that PAD-treatment inhibits the integrin-mediated TGF-β activation since PAD-treatment decreased the binding of integrin αVβ6 ectodomain as well as integrin-mediated spreading of MG-63 and HaCaT cells to β1-latency associated peptide (TGF-β1 LAP). The citrullination of the RGD site, an important integrin recognition motif, was confirmed by mass spectrometry. Furthermore, the citrullination of active TGF-β1 inhibited its binding to recombinant TGF-β receptor II, and prevented its ability to activate TGF-β signaling. Thus, extracellular PAD activity can affect the function of ECM-associated growth factors by different mechanisms. Importantly, the citrullination of both latent and active TGF-β has the potency to regulate the inflammatory process.

  6. Changes in extracellular matrix composition regulate cyclooxygenase-2 expression in human mesangial cells.

    PubMed

    Alique, Matilde; Calleros, Laura; Luengo, Alicia; Griera, Mercedes; Iñiguez, Miguel Ángel; Punzón, Carmen; Fresno, Manuel; Rodríguez-Puyol, Manuel; Rodríguez-Puyol, Diego

    2011-04-01

    Glomerular diseases are characterized by a sustained synthesis and accumulation of abnormal extracellular matrix proteins, such as collagen type I. The extracellular matrix transmits information to cells through interactions with membrane components, which directly activate many intracellular signaling events. Moreover, accumulating evidence suggests that eicosanoids derived from cyclooxygenase (COX)-2 participate in a number of pathological processes in immune-mediated renal diseases, and it is known that protein kinase B (AKT) may act through different transcription factors in the regulation of the COX-2 promoter. The present results show that progressive accumulation of collagen I in the extracellular medium induces a significant increase of COX-2 expression in human mesangial cells, resulting in an enhancement in PGE(2) production. COX-2 overexpression is due to increased COX-2 mRNA levels. The study of the mechanism implicated in COX-2 upregulation by collagen I showed focal adhesion kinase (FAK) activation. Furthermore, we observed that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by collagen I and collagen I-induced COX-2 overexpression was abolished by PI3K and AKT inhibitors. Additionally, we showed that the cAMP response element (CRE) transcription factor is implicated. Finally, we studied COX-2 expression in an animal model, N(G)-nitro-l-arginine methyl ester hypertensive rats. In renal tissue and vascular walls, COX-2 and collagen type I content were upregulated. In summary, our results provide evidence that collagen type I increases COX-2 expression via the FAK/PI3K/AKT/cAMP response element binding protein signaling pathway.

  7. High resolution three-dimensional reconstruction of fibrotic skeletal muscle extracellular matrix.

    PubMed

    Gillies, Allison R; Chapman, Mark A; Bushong, Eric A; Deerinck, Thomas J; Ellisman, Mark H; Lieber, Richard L

    2017-02-15

    Fibrosis occurs secondary to many skeletal muscle diseases and injuries, and can alter muscle function. It is unknown how collagen, the most abundant extracellular structural protein, alters its organization during fibrosis. Quantitative and qualitative high-magnification electron microscopy shows that collagen is organized into perimysial cables which increase in number in a model of fibrosis, and cables have unique interactions with collagen-producing cells. Fibrotic muscles are stiffer and have a higher concentration of collagen-producing cells. These results improve our understanding of the organization of fibrotic skeletal muscle extracellular matrix and identify novel structures that might be targeted by antifibrotic therapy. Skeletal muscle extracellular matrix (ECM) structure and organization are not well understood, yet the ECM plays an important role in normal tissue homeostasis and disease processes. Fibrosis is common to many muscle diseases and is typically quantified based on an increase in ECM collagen. Through the use of multiple imaging modalities and quantitative stereology, we describe the structure and composition of wild-type and fibrotic ECM, we show that collagen in the ECM is organized into large bundles of fibrils, or collagen cables, and the number of these cables (but not their size) increases in desmin knockout muscle (a fibrosis model). The increase in cable number is accompanied by increased muscle stiffness and an increase in the number of collagen producing cells. Unique interactions between ECM cells and collagen cables were also observed and reconstructed by serial block face scanning electron microscopy. These results demonstrate that the muscle ECM is more highly organized than previously reported. Therapeutic strategies for skeletal muscle fibrosis should consider the organization of the ECM to target the structures and cells contributing to fibrotic muscle function. © 2016 Rehabilitation Institute of Chicago. The Journal of

  8. Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

    PubMed

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-06-29

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

  9. Adipose Tissue and Extracellular Matrix Development by Injectable Decellularized Adipose Matrix Loaded with Basic Fibroblast Growth Factor.

    PubMed

    Zhang, Shipin; Lu, Qiqi; Cao, Tong; Toh, Wei Seong

    2016-04-01

    There is a significant need for soft-tissue replacements in the field of reconstructive surgery. Decellularized adipose tissues were heparin crosslinked and loaded with basic fibroblast growth factor (bFGF). This injectable system was evaluated for its adipogenic and angiogenic capabilities for in vivo adipose tissue regeneration. Decellularized adipose tissues were harvested from the inguinal fat pads of C57BL/6J mice, minced, and heparinized before being loaded with bFGF. Decellularized adipose tissues without bFGF served as a control. In vivo adipose neotissue formation, neovascularization, and volume stability were evaluated over a period of 12 weeks. After 6 or 12 weeks, mice were killed and the newly formed adipose tissues, together with the contralateral endogenous adipose tissues, were harvested for gross, volumetric, histologic, and immunohistochemical analysis. Decellularized adipose tissues that were heparinized and loaded with bFGF induced significant de novo adipose neotissue formation, with progressive tissue growth and neovascularization from 6 to 12 weeks. The adipose neotissues exhibited mature adipose morphology and extracellular matrix that closely resembled that of the endogenous adipose tissue. In contrast, decellularized adipose tissues without bFGF induced limited adipose neotissue formation and were completely resorbed by the end of 12 weeks. This study demonstrates the high efficiency of heparinized decellularized adipose tissue matrix loaded with bFGF in promoting adipose neotissue formation and neovascularization with long-term volume stability.

  10. Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

    PubMed Central

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-01-01

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilag