WorldWideScience

Sample records for human extracellular matrix

  1. Bioengineering Human Myocardium on Native Extracellular Matrix

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  3. Factors Involved in Extracellular Matrix Turnover in Human Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Gregori Casals

    2013-11-01

    Full Text Available Background: The molecular mechanisms by which myocardial ischemia translates into ventricular remodeling remain unclear. Methods: We investigated whether hypoxia and proinflammatory cytokines are specific inducers of remodeling signals in an in vitro model of cultured adult human ventricular myocytes (AC16 cells. Results:Hypoxia modified the ratio of matrix remodeling factors by increasing the aminoterminal propeptide of type III procollagen (PIIINP and reducing tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1 secretion in AC16 cells. These effects, however, were not associated with either modifications in expression of matrix metalloproteinase type 2, collagen-I or metalloproteinase activity. Hypoxia does, actually increase the production of the cardiac antifibrogenic growth factors, Apelin and VEGF, through an Hypoxia Inducible Factor type 1-dependent mechanism. Concerning proinflammatory signaling pathways, IL1β emerged as a powerful inducer of matrix turnover, since it significantly enhanced PIIINP, TIMP-1 and hyaluronic acid production and increased metalloproteinase activity. In contrast, TNFα did not modify matrix turnover but markedly induced the production of Apelin and VEGF. Conclusion: Hypoxia and increased TNFα activity likely exert cardioprotective actions by activating the cardiac antifibrogenic factors Apelin and VEGF. In contrast, IL1β is a strong promoter of interstitial collagen remodeling that may contribute to ventricular dilation and heart failure in the ischemic myocardium.

  4. Degenerated human intervertebral discs contain autoantibodies against extracellular matrix proteins

    Directory of Open Access Journals (Sweden)

    S Capossela

    2014-04-01

    Full Text Available Degeneration of intervertebral discs (IVDs is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.

  5. Degenerated human intervertebral discs contain autoantibodies against extracellular matrix proteins.

    Science.gov (United States)

    Capossela, S; Schläfli, P; Bertolo, A; Janner, T; Stadler, B M; Pötzel, T; Baur, M; Stoyanov, J V

    2014-04-04

    Degeneration of intervertebral discs (IVDs) is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP) to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.

  6. Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution.

    Directory of Open Access Journals (Sweden)

    Andrea Baiocchini

    Full Text Available 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.

  7. Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques

    DEFF Research Database (Denmark)

    Woods, Alan A; Linton, Stuart M; Davies, Michael Jonathan

    2003-01-01

    for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions......Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has...... by activated monocytes (and possibly macrophages) and is a highly basic protein, it would be expected to associate with polyanions such as the glycosaminoglycans of the extracellular matrix, and might result in damage being localized at such sites. In this study proteins extracted from extracellular matrix...

  8. Development of biomimetic nanocomposites as bone extracellular matrix for human osteoblastic cells.

    Science.gov (United States)

    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.

  9. Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

    Science.gov (United States)

    Tjin, Monica Suryana; Chua, Alvin Wen Choong; Ma, Dong Rui; Lee, Seng Teik; Fong, Eileen

    2014-08-01

    Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

  10. Extracellular matrix structure.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  12. Host-Parasite Interaction: Parasite-Derived and -Induced Proteases That Degrade Human Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Carolina Piña-Vázquez

    2012-01-01

    Full Text Available 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.

  13. Visualization of extracellular matrix components within sectioned Salmonella biofilms on the surface of human gallstones.

    Directory of Open Access Journals (Sweden)

    Joanna M Marshall

    Full Text Available Chronic carriage of Salmonella Typhi is mediated primarily through the formation of bacterial biofilms on the surface of cholesterol gallstones. Biofilms, by definition, involve the formation of a bacterial community encased within a protective macromolecular matrix. Previous work has demonstrated the composition of the biofilm matrix to be complex and highly variable in response to altered environmental conditions. Although known to play an important role in bacterial persistence in a variety of contexts, the Salmonella biofilm matrix remains largely uncharacterized under physiological conditions. Initial attempts to study matrix components and architecture of the biofilm matrix on gallstone surfaces were hindered by the auto-fluorescence of cholesterol. In this work we describe a method for sectioning and direct visualization of extracellular matrix components of the Salmonella biofilm on the surface of human cholesterol gallstones and provide a description of the major matrix components observed therein. Confocal micrographs revealed robust biofilm formation, characterized by abundant but highly heterogeneous expression of polysaccharides such as LPS, Vi and O-antigen capsule. CsgA was not observed in the biofilm matrix and flagellar expression was tightly restricted to the biofilm-cholesterol interface. Images also revealed the presence of preexisting Enterobacteriaceae encased within the structure of the gallstone. These results demonstrate the use and feasibility of this method while highlighting the importance of studying the native architecture of the gallstone biofilm. A better understanding of the contribution of individual matrix components to the overall biofilm structure will facilitate the development of more effective and specific methods to disrupt these bacterial communities.

  14. Visualization of Extracellular Matrix Components within Sectioned Salmonella Biofilms on the Surface of Human Gallstones

    Science.gov (United States)

    Marshall, Joanna M.; Flechtner, Alan D.; La Perle, Krista M.; Gunn, John S.

    2014-01-01

    Chronic carriage of Salmonella Typhi is mediated primarily through the formation of bacterial biofilms on the surface of cholesterol gallstones. Biofilms, by definition, involve the formation of a bacterial community encased within a protective macromolecular matrix. Previous work has demonstrated the composition of the biofilm matrix to be complex and highly variable in response to altered environmental conditions. Although known to play an important role in bacterial persistence in a variety of contexts, the Salmonella biofilm matrix remains largely uncharacterized under physiological conditions. Initial attempts to study matrix components and architecture of the biofilm matrix on gallstone surfaces were hindered by the auto-fluorescence of cholesterol. In this work we describe a method for sectioning and direct visualization of extracellular matrix components of the Salmonella biofilm on the surface of human cholesterol gallstones and provide a description of the major matrix components observed therein. Confocal micrographs revealed robust biofilm formation, characterized by abundant but highly heterogeneous expression of polysaccharides such as LPS, Vi and O-antigen capsule. CsgA was not observed in the biofilm matrix and flagellar expression was tightly restricted to the biofilm-cholesterol interface. Images also revealed the presence of preexisting Enterobacteriaceae encased within the structure of the gallstone. These results demonstrate the use and feasibility of this method while highlighting the importance of studying the native architecture of the gallstone biofilm. A better understanding of the contribution of individual matrix components to the overall biofilm structure will facilitate the development of more effective and specific methods to disrupt these bacterial communities. PMID:24551241

  15. Extracellular Matrix Proteins

    Directory of Open Access Journals (Sweden)

    Linda Christian Carrijo-Carvalho

    2012-01-01

    Full Text Available Lipocalin family members have been implicated in development, regeneration, and pathological processes, but their roles are unclear. Interestingly, these proteins are found abundant in the venom of the Lonomia obliqua caterpillar. Lipocalins are β-barrel proteins, which have three conserved motifs in their amino acid sequence. One of these motifs was shown to be a sequence signature involved in cell modulation. The aim of this study is to investigate the effects of a synthetic peptide comprising the lipocalin sequence motif in fibroblasts. This peptide suppressed caspase 3 activity and upregulated Bcl-2 and Ki-67, but did not interfere with GPCR calcium mobilization. Fibroblast responses also involved increased expression of proinflammatory mediators. Increase of extracellular matrix proteins, such as collagen, fibronectin, and tenascin, was observed. Increase in collagen content was also observed in vivo. Results indicate that modulation effects displayed by lipocalins through this sequence motif involve cell survival, extracellular matrix remodeling, and cytokine signaling. Such effects can be related to the lipocalin roles in disease, development, and tissue repair.

  16. Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1.

    Science.gov (United States)

    Mewhort, Holly E M; Lipon, Brodie D; Svystonyuk, Daniyil A; Teng, Guoqi; Guzzardi, David G; Silva, Claudia; Yong, V Wee; Fedak, Paul W M

    2016-03-15

    Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1. These data implicate inflammation as a potential driver of cardiac fibrosis.

  17. Assembly of fibronectin into the extracellular matrix of early and late passage human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Mann, D.M.

    1987-01-01

    The specific binding of soluble /sup 125/I-human plasma fibronectin (/sup 125/I-HFN-P) to confluent cultures of early and late passage human skin fibroblasts was investigated. Previous studies HFN-P bound to fibroblast cell layers indicated that HNF-P was present in the cultures in two separate pools, distinguishable on the basis of their solubility in 1% deoxycholate. Examination of the kinetics of /sup 125/I-HFN-P binding to Pool I of early and late passage cultures revealed that both cultures required 2-4 h to approach steady-state conditions. Other kinetic studies showed that the rates of low of /sup 125/I-HFN-P from either Pool I or Pool II were similar for both cultures. Further, Scatchard analysis revealed a single class of Pool I binding sites with apparent dissociation constants (K/sub d/) of 5.3 x 10/sup -8/M (early passage) and 4.2 x 10/sup -8/M (late passage). These results indicate that early and late passage cultures of human fibroblasts exhibit differences in the number of cell surface biding sites for soluble fibronectin, and in the extent to which they incorporate soluble fibronectin into the extracellular matrix. Parameters which affect the fibronectin matrix assembly system of human skin fibroblasts were also examined. In addition, several monoclonal anti-fibronectin antibodies were characterized and developed as experimental probes for fibronectin structure and function.

  18. Changes in extracellular matrix composition regulate cyclooxygenase-2 expression in human mesangial cells.

    Science.gov (United States)

    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.

  19. Establishment and characterization of human engineered cells stably expressing large extracellular matrix proteins.

    Science.gov (United States)

    Kwon, Daekee; Kang, Gwang-Sik; Han, Dong Keun; Park, Kwideok; Kim, Jae-Hwan; Lee, Soo-Hong

    2014-01-01

    Commercially available extracellular matrix (ECM) hydrogel-coated culture plates have been used to study the relationship between the ECM microenvironment and stem cell behavior. However, it is unclear whether ECM-coated dishes mimic the natural ECM microenvironment because the architecture of the ECM is constructed of randomly distributed fibers. The purpose of this study was the production and confirmation of human engineered cell lines stably expressing large ECM proteins such as collagen I/II and fibronectin. First, large (over 10 kb) ECM vectors encoding human collagen I/II and fibronectin were constructed and the circular vectors were linearized. Second, the linear ECM vectors were introduced into immortalized human embryonic kidney cells using various transfection methods. The polyethylenimine and liposome methods showed higher efficiencies than electroporation for transfection of these large vectors. Third, human ECM engineered cells were established by stable integration of the vector into the genomic DNA and resulted in stable overexpression of mRNA and proteins. In summary, human engineered cell lines stably expressing large ECM proteins such as human collagen I/II and fibronectin were successfully prepared, and secretion of the ECM components into the surrounding environment was confirmed by immunocytochemistry. Thus, human ECM engineered cells naturally secreting ECM components could be valuable for studying the relationship between the native ECM microenvironment and stem cell behavior.

  20. Human hamstring tenocytes survive when seeded into a decellularized porcine Achilles tendon extracellular matrix.

    Science.gov (United States)

    Lohan, Anke; Stoll, Christiane; Albrecht, Marit; Denner, Andreas; John, Thilo; Krüger, Kay; Ertel, Wolfgang; Schulze-Tanzil, Gundula

    2013-01-01

    Tendon ruptures and defects remain major orthopaedic challenges. Tendon healing is a time-consuming process, which results in scar tissue with an altered biomechanical competence. Using a xenogeneic tendon extracellular matrix (ECM) as a natural scaffold, which can be reseeded with autologous human tenocytes, might be a promising approach to reconstruct damaged tendons. For this purpose, the porcine Achilles (AS) tendons serving as a scaffold were histologically characterized in comparison to human cell donor tendons. AS tendons were decellularized and then reseeded with primary human hamstring tenocytes using cell centrifuging, rotating culture and cell injection techniques. Vitality testing, histology and glycosaminoglycan/DNA quantifications were performed to document the success of tendon reseeding. Porcine AS tendons were characterized by a higher cell and sulfated glycosaminoglycan content than human cell donor tendons. Complete decellularization could be achieved, but led to a wash out of sulfated glycosaminoglycans. Nevertheless, porcine tendon could be recellularized with vital human tenocytes. The recellularization led to a slight increase in cell number compared to the native tendon and some glycosaminoglycan recovery. This study indicates that porcine tendon can be de- and recellularized using adult human tenocytes. Future work should optimize cell distribution within the recellularized tendon ECM and consider tendon- and donor species-dependent differences.

  1. Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells

    DEFF Research Database (Denmark)

    Oberwallner, Barbara; Brodarac, Andreja; Anić, Petra;

    2015-01-01

    lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell......OBJECTIVES: Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation...... that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure. METHODS: Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic...

  2. Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions.

    Science.gov (United States)

    Karkampouna, Sofia; Kloen, Peter; Obdeijn, Miryam C; Riester, Scott M; van Wijnen, Andre J; Kruithof-de Julio, Marianna

    2015-01-01

    Organ fibrosis or "scarring" is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic. To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first "organ" culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

  3. Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane

    Science.gov (United States)

    Lei, Jennifer; Priddy, Lauren B.; Lim, Jeremy J.; Massee, Michelle; Koob, Thomas J.

    2017-01-01

    Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION® Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek® activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair. PMID:28224047

  4. Extracellular Matrix Hydrogel Derived from Human Umbilical Cord as a Scaffold for Neural Tissue Repair and Its Comparison with Extracellular Matrix from Porcine Tissues.

    Science.gov (United States)

    Kočí, Zuzana; Výborný, Karel; Dubišová, Jana; Vacková, Irena; Jäger, Aleš; Lunov, Oleg; Jiráková, Klára; Kubinová, Šárka

    2017-06-01

    Extracellular matrix (ECM) hydrogels prepared by tissue decellularization have been reported as natural injectable materials suitable for neural tissue repair. In this study, we prepared ECM hydrogel derived from human umbilical cord (UC) and evaluated its composition and mechanical and biological properties in comparison with the previously described ECM hydrogels derived from porcine urinary bladder (UB), brain, and spinal cord. The ECM hydrogels did not differ from each other in the concentration of collagen, while the highest content of glycosaminoglycans as well as the shortest gelation time was found for UC-ECM. The elastic modulus was then found to be the highest for UB-ECM. In spite of a different origin, topography, and composition, all ECM hydrogels similarly promoted the migration of human mesenchymal stem cells (MSCs) and differentiation of neural stem cells, as well as axonal outgrowth in vitro. However, only UC-ECM significantly improved proliferation of tissue-specific UC-derived MSCs when compared with the other ECMs. Injection of UC-ECM hydrogels into a photothrombotic cortical ischemic lesion in rats proved its in vivo gelation and infiltration with host macrophages. In summary, this study proposes UC-ECM hydrogel as an easily accessible biomaterial of human origin, which has the potential for neural as well as other soft tissue reconstruction.

  5. Photobiomodulation on human annulus fibrosus cells during the intervertebral disk degeneration: extracellular matrix-modifying enzymes.

    Science.gov (United States)

    Hwang, Min Ho; Kim, Kyoung Soo; Yoo, Chang Min; Shin, Jae Hee; Nam, Hyo Geun; Jeong, Jin Su; Kim, Joo Han; Lee, Kwang Ho; Choi, Hyuk

    2016-05-01

    Destruction of extracellular matrix (ECM) leads to degeneration of the intervertebral disk (IVD), which is a major contributor to many spine disorders. IVD degeneration is induced by pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), which are secreted by immune cells, including macrophages and neutrophils. The cytokines modulate ECM-modifying enzymes such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human annulus fibrosus (AF) cells. The resulting imbalance in catabolic and anabolic enzymes can cause generalized back, neck, and low back pain (LBP). Photobiomodulation (PBM) is known to regulate inflammatory responses and wound healing. The aim of this study was to mimic the degenerative IVD microenvironment, and to investigate the effect of a variety of PBM conditions (wavelength: 635, 525, and 470 nm; energy density: 16, 32, and 64 J/cm(2)) on the production of ECM-modifying-enzymes by AF cells under degenerative conditions induced by macrophage-conditioned medium (MCM), which contains pro-inflammatory cytokines such as TNF-α and IL-β secreted by macrophage during the development of intervertebral disk inflammation. We showed that the MCM-stimulated AF cells express imbalanced ratios of TIMPs (TIMP-1 and TIMP-2) and MMPs (MMP-1 and MMP-3). PBM selectively modulated the production of ECM-modifying enzymes in AF cells. These results suggest that PBM can be a therapeutic tool for degenerative IVD disorders.

  6. Scanning Electron Microscopic Examination of the Extracellular Matrix in the Decellularized Mouse and Human Cochlea.

    Science.gov (United States)

    Santi, Peter A; Aldaya, Robair; Brown, Alec; Johnson, Shane; Stromback, Tyler; Cureoglu, Sebahattin; Rask-Andersen, Helge

    2016-06-01

    Decellularized tissues have been used to investigate the extracellular matrix (ECM) in a number of different tissues and species. Santi and Johnson JARO 14:3-15 (2013) first described the decellularized inner ear in the mouse, rat, and human using scanning thin-sheet laser imaging microscopy (sTSLIM). The purpose of the present investigation is to examine decellularized cochleas in the mouse and human at higher resolution using scanning electron microscopy (SEM). Fresh cochleas were harvested and decellularized using detergent extraction methods. Following decellularization, the ECM of the bone, basilar membrane, spiral limbus, and ligament remained, and all of the cells were removed from the cochlea. A number of similarities and differences in the ECM of the mouse and human were observed. A novel, spirally directed structure was present on the basilar membrane and is located at the border between Hensen and Boettcher cells. These septa-like structures formed a single row in the mouse and multiple rows in the human. The basal lamina of the stria vascularis capillaries was present and appeared thicker in the human compared with the mouse. In the mouse, numerous openings beneath the spiral prominence that previously housed the root processes of the external sulcus cells were observed but in the human there was only a single row of openings. These and other anatomical differences in the ECM between the mouse and human may reflect functional differences and/or be due to aging; however, decellularized cochleas provide a new way to examine the cochlear ECM and reveal new observations.

  7. Interactions of human tenascin-X domains with dermal extracellular matrix molecules.

    NARCIS (Netherlands)

    Egging, D.; Berkmortel, F. van den; Taylor, G.; Bristow, J.; Schalkwijk, J.

    2007-01-01

    Tenascin-X (TNX) is a large 450 kDa extracellular matrix protein expressed in a variety of tissues including skin, joints and blood vessels. Deficiency of TNX causes a recessive form of Ehlers-Danlos syndrome characterized by joint hypermobility, skin fragility and hyperextensible skin. Skin of TNX

  8. Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions

    NARCIS (Netherlands)

    D.C. MacLeod (Donald); B.H. Strauss (Bradley); J. Escaned (Javier); V.A.W.M. Umans (Victor); R-J. van Suylen (Robert-Jan); A. Verkerk (Anton); P.J. de Feyter (Pim); P.W.J.C. Serruys (Patrick); M. de Jong (Marcel)

    1994-01-01

    textabstractOBJECTIVES. The purpose of this study was to examine the proliferative capacity and extracellular matrix synthesis of human coronary plaque cells in vitro. BACKGROUND. Common to both primary atherosclerosis and restenosis are vascular smooth muscle cell proliferation and production of ex

  9. Impact of acetaminophen consumption and resistance exercise on extracellular matrix gene expression in human skeletal muscle.

    Science.gov (United States)

    Patel, Shivam H; D'Lugos, Andrew C; Eldon, Erica R; Curtis, Donald; Dickinson, Jared M; Carroll, Chad C

    2017-07-01

    Acetaminophen (APAP) given during chronic exercise reduces skeletal muscle collagen and cross-linking in rats. We propose that the effect of APAP on muscle extracellular matrix (ECM) may, in part, be mediated by dysregulation of the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). The purpose of this study was to evaluate the impact of APAP consumption during acute resistance exercise (RE) on several regulators of the ECM in human skeletal muscle. In a double-blinded, placebo-controlled, randomized crossover design, recreationally active men (n = 8, 25 ± 2 yr) performed two trials of knee extension. Placebo (PLA) or APAP (1,000 mg/6 h) was given for 24 h before and immediately following RE. Vastus lateralis biopsies were taken at baseline and 1 and 3 h post-RE. Quantitative RT-PCR was used to determine differences in mRNA expression. MMP-2, type I collagen, and type III collagen mRNA expression was not altered by exercise or APAP (P > 0.05). When compared with PLA, TIMP-1 expression was lower at 1 h post-RE during APAP conditions but greater than PLA at 3 h post-RE (P 0.05). Phosphorylation of ERK1/2 and p38-MAPK increased (P < 0.05) with RE but was not influenced by APAP. Our findings do not support our hypothesis and suggest that short-term APAP consumption before RE has a small impact on the measured ECM molecules in human skeletal muscle following acute RE. Copyright © 2017 the American Physiological Society.

  10. Biocompatibility of pure titanium modified by human endothelial cell-derived extracellular matrix

    Energy Technology Data Exchange (ETDEWEB)

    Xue Xiaoqing [Key Laboratory of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Wang Jin, E-mail: jinxxwang@263.net [Key Laboratory of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Zhu Ying; Tu Qiufen; Huang Nan [Key Laboratory of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China)

    2010-04-01

    Extracellular matrix (ECM) used to modify biomaterial surface is a promising method for improving cardiovascular material hemocompatibility. In the present work, human umbilical vein endothelial cells (HUVECs) are cultured and native ECM is obtained on pure titanium surface. Fourier infrared spectrum (FTIR) test proves the existence of amide I and amide II band on the modified titanium surface. X-ray photoelectron spectroscopy (XPS) further confirms the chemical composition and binding types of the ECM proteins on the titanium substrate. The results of light microscopy and atomic force microscopy (AFM) exhibit the morphology of HUVEC derived ECM. There are higher water contact angles on the ECM modified samples. Furthermore, some ECM components, including fibronectin (FN), laminin (LN) and type IV collagen (IV-COL) are presented on ECM-covered titanium surface by immunofluorescence staining. The biological behavior of cultured HUVECs and adherent platelets on different samples are investigated by in vitro HUVECs culture and platelet adhesion. Cells exhibit better morphology and their proliferation ability greatly improve on the ECM-covered titanium. At the same time, the platelet adhesion and spreading are inhibited on ECM-covered titanium surface. These investigations demonstrate that ECM produced by HUVECs cannot only improve adhesion and proliferation ability of endothelial cell but also inhibit adhesion and activation of platelets. Thus, the approach described here may provide a basis for preparation of modified surface in cardiovascular implants application.

  11. Urine proteomes of healthy aging humans reveal extracellular matrix (ECM) alterations and immune system dysfunction.

    Science.gov (United States)

    Bakun, M; Senatorski, G; Rubel, T; Lukasik, A; Zielenkiewicz, P; Dadlez, M; Paczek, L

    2014-02-01

    Aging is a complex physiological process that poses considerable conundrums to rapidly aging societies. For example, the risk of dying from cardiovascular diseases and/or cancer steadily declines for people after their 60s, and other causes of death predominate for seniors older than 80 years of age. Thus, physiological aging presents numerous unanswered questions, particularly with regard to changing metabolic patterns. Urine proteomics analysis is becoming a non-invasive and reproducible diagnostic method. We investigated the urine proteomes in healthy elderly people to determine which metabolic processes were weakened or strengthened in aging humans. Urine samples from 37 healthy volunteers aged 19-90 years (19 men, 18 women) were analyzed for protein expression by liquid chromatography-tandem mass spectrometry. This generated a list of 19 proteins that were differentially expressed in different age groups (young, intermediate, and old age). In particular, the oldest group showed protein changes reflective of altered extracellular matrix turnover and declining immune function, in which changes corresponded to reported changes in cardiovascular tissue remodeling and immune disorders in the elderly. Thus, urinary proteome changes in the elderly appear to reflect the physiological processes of aging and are particularly clearly represented in the circulatory and immune systems. Detailed identification of "protein trails" creates a more global picture of metabolic changes that occur in the elderly.

  12. In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins.

    Science.gov (United States)

    Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Khang, Gilson; Soker, Shay

    2013-02-01

    The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation.

  13. Distribution volumes of macromolecules in human ovarian and endometrial cancers--effects of extracellular matrix structure.

    Science.gov (United States)

    Haslene-Hox, Hanne; Oveland, Eystein; Woie, Kathrine; Salvesen, Helga B; Tenstad, Olav; Wiig, Helge

    2015-01-01

    Elements of the extracellular matrix (ECM), notably collagen and glucosaminoglycans, will restrict part of the space available for soluble macromolecules simply because the molecules cannot occupy the same space. This phenomenon may influence macromolecular drug uptake. To study the influence of steric and charge effects of the ECM on the distribution volumes of macromolecules in human healthy and malignant gynecologic tissues we used as probes 15 abundant plasma proteins quantified by high-resolution mass spectrometry. The available distribution volume (VA) of albumin was increased in ovarian carcinoma compared with healthy ovarian tissue. Furthermore, VA of plasma proteins between 40 and 190 kDa decreased with size for endometrial carcinoma and healthy ovarian tissue, but was independent of molecular weight for the ovarian carcinomas. An effect of charge on distribution volume was only found in healthy ovaries, which had lower hydration and high collagen content, indicating that a condensed interstitium increases the influence of negative charges. A number of earlier suggested biomarker candidates were detected in increased amounts in malignant tissue, e.g., stathmin and spindlin-1, showing that interstitial fluid, even when unfractionated, can be a valuable source for tissue-specific proteins. We demonstrate that the distribution of abundant plasma proteins in the interstitium can be elucidated by mass spectrometry methods and depends markedly on hydration and ECM structure. Our data can be used in modeling of drug uptake, and give indications on ECM components to be targeted to increase the uptake of macromolecular substances.

  14. IAPP modulates cellular autophagy, apoptosis, and extracellular matrix metabolism in human intervertebral disc cells

    Science.gov (United States)

    Wu, Xinghuo; Song, Yu; Liu, Wei; Wang, Kun; Gao, Yong; Li, Shuai; Duan, Zhenfeng; Shao, Zengwu; Yang, Shuhua; Yang, Cao

    2017-01-01

    The pathogenic process of intervertebral disc degeneration (IDD) is characterized by imbalance in the extracellular matrix (ECM) metabolism. Nucleus pulposus (NP) cells have important roles in maintaining the proper structure and tissue homeostasis of disc ECM. These cells need adequate supply of glucose and oxygen. Islet amyloid polypeptide (IAPP) exerts its biological effects by regulating glucose metabolism. The purpose of this study was to investigate the expression of IAPP in degenerated IVD tissue, and IAPP modulation of ECM metabolism in human NP cells, especially the crosstalk mechanism between apoptosis and autophagy in these cells. We found that the expression of IAPP and Calcr-RAMP decreased considerably during IDD progression, along with the decrease in the expression of AG, BG, and Col2A1. Induction of IAPP in NP cells by transfection with pLV-IAPP enhanced the synthesis of aggrecan and Col2A1 and attenuated the expression of pro-inflammatory factors, tumor necrosis factor (TNF)-α, and interleukin (IL)-1. Upregulation of IAPP also affected the expression of the catabolic markers—matrix metalloproteinases (MMPs) 3, 9 and 13 and ADAMTS 4 and 5. Downregulation of IAPP by siRNA inhibited the expression of anabolic genes but increased the expression of catabolic genes and inflammatory factors. The expressions of autophagic and apoptotic markers in NP cells transfected with pLV-IAPP were upregulated, including BECLIN1, ATG5, ATG7, LC3 II/I and Bcl-2, while significantly increase in the expression of Bax and Caspase-3 in NP cells transfected with pLV-siIAPP. Mechanistically, PI3K/AKT-mTOR and p38/JNK MAPK signal pathways were involved. We propose that IAPP might play a pivotal role in the development of IDD, by regulating ECM metabolism and controlling the crosstalk between apoptosis and autophagy in NP, thus potentially offering a novel therapeutic approach to the treatment of IDD.

  15. Silicon-based quantum dots induce inflammation in human lung cells and disrupt extracellular matrix homeostasis.

    Science.gov (United States)

    Stan, Miruna-Silvia; Sima, Cornelia; Cinteza, Ludmila Otilia; Dinischiotu, Anca

    2015-08-01

    Quantum dots (QDs) are nanocrystalline semiconductor materials that have been tested for biological applications such as cancer therapy, cellular imaging and drug delivery, despite the serious lack of information of their effects on mammalian cells. The present study aimed to evaluate the potential of Si/SiO2 QDs to induce an inflammatory response in MRC-5 human lung fibroblasts. Cells were exposed to different concentrations of Si/SiO2 QDs (25-200 μg·mL(-1)) for 24, 48, 72 and 96 h. The results obtained showed that uptake of QDs was dependent on biocorona formation and the stability of nanoparticles in various biological media (minimum essential medium without or with 10% fetal bovine serum). The cell membrane damage indicated by the increase in lactate dehydrogenase release after exposure to QDs was dose- and time-dependent. The level of lysosomes increased proportionally with the concentration of QDs, whereas an accumulation of autophagosomes was also observed. Cellular morphology was affected, as shown by the disruption of actin filaments. The enhanced release of nitric oxide and the increase in interleukin-6 and interleukin-8 protein expression suggested that nanoparticles triggered an inflammatory response in MRC-5 cells. QDs decreased the protein expression and enzymatic activity of matrix metalloproteinase (MMP)-2 and MMP-9 and also MMP-1 caseinase activity, whereas the protein levels of MMP-1 and tissue inhibitor of metalloproteinase-1 increased. The present study reveals for the first time that silicon-based QDs are able to generate inflammation in lung cells and cause an imbalance in extracellular matrix turnover through a differential regulation of MMPs and tissue inhibitor of metalloproteinase-1 protein expression.

  16. CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells.

    Directory of Open Access Journals (Sweden)

    Elena P Moiseeva

    Full Text Available CADM1 is a major receptor for the adhesion of mast cells (MCs to fibroblasts, human airway smooth muscle cells (HASMCs and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM. Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

  17. Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta.

    Science.gov (United States)

    Watson, Chris J; Phelan, Dermot; Collier, Patrick; Horgan, Stephen; Glezeva, Nadia; Cooke, Gordon; Xu, Maojia; Ledwidge, Mark; McDonald, Kenneth; Baugh, John A

    2014-06-01

    Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

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

    Science.gov (United States)

    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.

  19. Histochemical evidence of osteoclastic degradation of extracellular matrix in osteolytic metastasis originating from human lung small carcinoma (SBC-5) cells.

    Science.gov (United States)

    Li, Minqi; Amizuka, Norio; Takeuchi, Kiichi; Freitas, Paulo H L; Kawano, Yoshiro; Hoshino, Masaaki; Oda, Kimimitsu; Nozawa-Inoue, Kayoko; Maeda, Takeyasu

    2006-02-01

    The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular

  20. PRDM14 Promotes the Migration of Human Non-small Cell Lung Cancer Through Extracellular Matrix Degradation in vitro

    Institute of Scientific and Technical Information of China (English)

    Hong-Xia Bi; Han-Bing Shi; Ting Zhang; Ge Cui

    2015-01-01

    Background:As a novel molecular markerof non-small cell lung cancer (NSCLC),PRDI-BF1 and RIZ homology domain containing protein 14 (PRDM 14) is over-expressed in NSCLC tumor tissues.Extracellular matrix degradation mediated by the balance between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) is one of the most important mechanism in lung cancer metastasis.This study aimed to determine if PRDM 14 promoted the migration of NSCLC cells through extracellular matrix degradation mediated by change of MMP/TIMP expression.Methods:The expression of PRDM14 was down-regulated in human cell line A 549 after transfection with lentiviral vector-mediated short-hairpin ribonucleic acids (shRNAs) which targeted the PRDM 14 promoter.Cellular migration ofshRNA-infected cells was detected by a scratch wound healing assay and transwell cell migration assay.Expression levels of MMP1,MMP2,TIMP1,and TIMP2 were measured by quantitative real-time polymerase chain reaction (RT-PCR).Results:Migration ofPRDM 14-shRNA-infected cells was significantly inhibited relative to control cells as measured by the scratch wound healing (P < 0.05) and transwell cell migration assays (P < 0.01).The expression of MMP1 in A549 cells infected by PRDM14-shRNA was down-regulated significantly (P < 0.01),whereas the expression of TIMPl and TIMP2 was up-regulated significantly (P < 0.01).Conclusions:PRDM 14 accelerates A549 cells migration in vitro through extracellular matrix degradation.PRDM 14 is considered as a potential therapeutic target in metastatic NSCLC.

  1. Changes in vascular extracellular matrix composition during decidual spiral arteriole remodeling in early human pregnancy.

    Science.gov (United States)

    Smith, Samantha D; Choudhury, Ruhul H; Matos, Patricia; Horn, James A; Lye, Stephen J; Dunk, Caroline E; Aplin, John D; Jones, Rebecca L; Harris, Lynda K

    2016-05-01

    Uterine spiral arteriole (SA) remodeling in early pregnancy involves a coordinated series of events including decidual immune cell recruitment, vascular cell disruption and loss, and colonization by placental-derived extravillous trophoblast (EVT). During this process, decidual SA are converted from narrow, muscular vessels into dilated channels lacking vasomotor control. We hypothesized that this extensive alteration in SA architecture must require significant reorganization and/or breakdown of the vascular extracellular matrix (ECM). First trimester decidua basalis (30 specimens) was immunostained to identify spiral arterioles undergoing trophoblast-independent and -dependent phases of remodeling. Serial sections were then immunostained for a panel of ECM markers, to examine changes in vascular ECM during the remodeling process. The initial stages of SA remodeling were characterized by loss of laminin, elastin, fibrillin, collagen types III, IV and VI from the basement membrane, vascular media and/or adventitia, and surrounding decidual stromal cells. Loss of ECM correlated with disruption and disorganization of vascular smooth muscle cells, and the majority of changes occurred prior to extensive colonization of the vessel wall by EVT. The final stages of SA remodeling, characterized by the arrival of EVT, were associated with the increased mural deposition of fibronectin and fibrinoid. This study provides the first detailed analysis of the spatial and temporal loss of ECM from the walls of remodeling decidual SA in early pregnancy.

  2. Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins

    Directory of Open Access Journals (Sweden)

    O.C. Lima

    1999-05-01

    Full Text Available The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate (poly-HEMA was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent. The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin was statistically significant (P<0.05 when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

  3. The Human Pancreas as a Source of Protolerogenic Extracellular Matrix Scaffold for a New-generation Bioartificial Endocrine Pancreas.

    Science.gov (United States)

    Peloso, Andrea; Urbani, Luca; Cravedi, Paolo; Katari, Ravi; Maghsoudlou, Panagiotis; Fallas, Mario Enrique Alvarez; Sordi, Valeria; Citro, Antonio; Purroy, Carolina; Niu, Guoguang; McQuilling, John P; Sittadjody, Sivanandane; Farney, Alan C; Iskandar, Samy S; Zambon, Joao P; Rogers, Jeffrey; Stratta, Robert J; Opara, Emmanuel C; Piemonti, Lorenzo; Furdui, Cristina M; Soker, Shay; De Coppi, Paolo; Orlando, Giuseppe

    2016-07-01

    Our study aims at producing acellular extracellular matrix scaffolds from the human pancreas (hpaECMs) as a first critical step toward the production of a new-generation, fully human-derived bioartificial endocrine pancreas. In this bioartificial endocrine pancreas, the hardware will be represented by hpaECMs, whereas the software will consist in the cellular compartment generated from patient's own cells. Extracellular matrix (ECM)-based scaffolds obtained through the decellularization of native organs have become the favored platform in the field of complex organ bioengineering. However, the paradigm is now switching from the porcine to the human model. To achieve our goal, human pancreata were decellularized with Triton-based solution and thoroughly characterized. Primary endpoints were complete cell and DNA clearance, preservation of ECM components, growth factors and stiffness, ability to induce angiogenesis, conservation of the framework of the innate vasculature, and immunogenicity. Secondary endpoint was hpaECMs' ability to sustain growth and function of human islet and human primary pancreatic endothelial cells. Results show that hpaECMs can be successfully and consistently produced from human pancreata and maintain their innate molecular and spatial framework and stiffness, and vital growth factors. Importantly, hpaECMs inhibit human naïve CD4 T-cell expansion in response to polyclonal stimuli by inducing their apoptosis and promoting their conversion into regulatory T cells. hpaECMs are cytocompatible and supportive of representative pancreatic cell types. We, therefore, conclude that hpaECMs has the potential to become an ideal platform for investigations aiming at the manufacturing of a regenerative medicine-inspired bioartificial endocrine pancreas.

  4. Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy

    Directory of Open Access Journals (Sweden)

    Olla Carlo

    2007-08-01

    Full Text Available Abstract Background The Down syndrome phenotype has been attributed to overexpression of chromosome 21 (Hsa21 genes. However, the expression profile of Hsa21 genes in trisomic human subjects as well as their effects on genes located on different chromosomes are largely unknown. Using oligonucleotide microarrays we compared the gene expression profiles of hearts of human fetuses with and without Hsa21 trisomy. Results Approximately half of the 15,000 genes examined (87 of the 168 genes on Hsa21 were expressed in the heart at 18–22 weeks of gestation. Hsa21 gene expression was globally upregulated 1.5 fold in trisomic samples. However, not all genes were equally dysregulated and 25 genes were not upregulated at all. Genes located on other chromosomes were also significantly dysregulated. Functional class scoring and gene set enrichment analyses of 473 genes, differentially expressed between trisomic and non-trisomic hearts, revealed downregulation of genes encoding mitochondrial enzymes and upregulation of genes encoding extracellular matrix proteins. There were no significant differences between trisomic fetuses with and without heart defects. Conclusion We conclude that dosage-dependent upregulation of Hsa21 genes causes dysregulation of the genes responsible for mitochondrial function and for the extracellular matrix organization in the fetal heart of trisomic subjects. These alterations might be harbingers of the heart defects associated with Hsa21 trisomy, which could be based on elusive mechanisms involving genetic variability, environmental factors and/or stochastic events.

  5. Oxidation modifies the structure and function of the extracellular matrix generated by human coronary artery endothelial cells.

    Science.gov (United States)

    Chuang, Christine Y; Degendorfer, Georg; Hammer, Astrid; Whitelock, John M; Malle, Ernst; Davies, Michael J

    2014-04-15

    ECM (extracellular matrix) materials, such as laminin, perlecan, type IV collagen and fibronectin, play a key role in determining the structure of the arterial wall and the properties of cells that interact with the ECM. The aim of the present study was to investigate the effect of peroxynitrous acid, an oxidant generated by activated macrophages, on the structure and function of the ECM laid down by HCAECs (human coronary artery endothelial cells) in vitro and in vivo. We show that exposure of HCAEC-derived native matrix components to peroxynitrous acid (but not decomposed oxidant) at concentrations >1 μM results in a loss of antibody recognition of perlecan, collagen IV, and cell-binding sites on laminin and fibronectin. Loss of recognition was accompanied by decreased HCAEC adhesion. Real-time PCR showed up-regulation of inflammation-associated genes, including MMP7 (matrix metalloproteinase 7) and MMP13, as well as down-regulation of the laminin α2 chain, in HCAECs cultured on peroxynitrous acid-treated matrix compared with native matrix. Immunohistochemical studies provided evidence of co-localization of laminin with 3-nitrotyrosine, a biomarker of peroxynitrous acid damage, in type II-III/IV human atherosclerotic lesions, consistent with matrix damage occurring during disease development in vivo. The results of the present study suggest a mechanism through which peroxynitrous acid modifies endothelial cell-derived native ECM proteins of the arterial basement membrane in atherosclerotic lesions. These changes to ECM and particularly perlecan and laminin may be important in inducing cellular dysfunction and contribute to atherogenesis.

  6. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

    Science.gov (United States)

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego

    2013-02-01

    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.

  7. Comparative adherence of Candida albicans and Candida dubliniensis to human buccal epithelial cells and extracellular matrix proteins.

    Science.gov (United States)

    Jordan, Rachael P C; Williams, David W; Moran, Gary P; Coleman, David C; Sullivan, Derek J

    2014-04-01

    Candida albicans and Candida dubliniensis are very closely related pathogenic yeast species. Despite their close relationship, C. albicans is a far more successful colonizer and pathogen of humans. The purpose of this study was to determine if the disparity in the virulence of the two species is attributed to differences in their ability to adhere to human buccal epithelial cells (BECs) and/or extracellular matrix proteins. When grown overnight at 30°C in yeast extract peptone dextrose, genotype 1 C. dubliniensis isolates were found to be significantly more adherent to human BECs than C. albicans or C. dubliniensis genotypes 2-4 (P albicans to human BECs was observed, and C. dubliniensis genotype 1 and C. albicans adhered to BECs in significantly greater numbers than the other C. dubliniensis genotypes (P albicans to type I and IV collagen, fibronectin, laminin, vitronectin, and proline-rich peptides. These data suggest that C. albicans is not more adherent to epithelial cells or matrix proteins than C. dubliniensis and therefore other factors must contribute to the greater levels of virulence exhibited by C. albicans.

  8. Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.

    Science.gov (United States)

    Bridgen, D T; Gilchrist, C L; Richardson, W J; Isaacs, R E; Brown, C R; Yang, K L; Chen, J; Setton, L A

    2013-10-01

    The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics.

  9. Influence of culture conditions and extracellular matrix alignment on human mesenchymal stem cells invasion into decellularized engineered tissues.

    Science.gov (United States)

    Weidenhamer, Nathan K; Moore, Dusty L; Lobo, Fluvio L; Klair, Nathaniel T; Tranquillo, Robert T

    2015-05-01

    The variables that influence the in vitro recellularization potential of decellularized engineered tissues, such as cell culture conditions and scaffold alignment, have yet to be explored. The goal of this work was to explore the influence of insulin and ascorbic acid and extracellular matrix (ECM) alignment on the recellularization of decellularized engineered tissue by human mesenchymal stem cells (hMSCs). Aligned and non-aligned tissues were created by specifying the geometry and associated mechanical constraints to fibroblast-mediated fibrin gel contraction and remodelling using circular and C-shaped moulds. Decellularized tissues (matrices) of the same alignment were created by decellularization with detergents. Ascorbic acid promoted the invasion of hMSCs into the matrices due to a stimulated increase in motility and proliferation. Invasion correlated with hyaluronic acid secretion, α-smooth muscle actin expression and decreased matrix thickness. Furthermore, hMSCs invasion into aligned and non-aligned matrices was not different, although there was a difference in cell orientation. Finally, we show that hMSCs on the matrix surface appear to differentiate toward a smooth muscle cell or myofibroblast phenotype with ascorbic acid treatment. These results inform the strategy of recellularizing decellularized engineered tissue with hMSCs.

  10. The mechanical properties of human adipose tissues and their relationships to the structure and composition of the extracellular matrix.

    Science.gov (United States)

    Alkhouli, Nadia; Mansfield, Jessica; Green, Ellen; Bell, James; Knight, Beatrice; Liversedge, Neil; Tham, Ji Chung; Welbourn, Richard; Shore, Angela C; Kos, Katarina; Winlove, C Peter

    2013-12-01

    Adipose tissue (AT) expansion in obesity is characterized by cellular growth and continuous extracellular matrix (ECM) remodeling with increased fibrillar collagen deposition. It is hypothesized that the matrix can inhibit cellular expansion and lipid storage. Therefore, it is important to fully characterize the ECM's biomechanical properties and its interactions with cells. In this study, we characterize and compare the mechanical properties of human subcutaneous and omental tissues, which have different physiological functions. AT was obtained from 44 subjects undergoing surgery. Force/extension and stress/relaxation data were obtained. The effects of osmotic challenge were measured to investigate the cellular contribution to tissue mechanics. Tissue structure and its response to tensile strain were determined using nonlinear microscopy. AT showed nonlinear stress/strain characteristics of up to a 30% strain. Comparing paired subcutaneous and omental samples (n = 19), the moduli were lower in subcutaneous: initial 1.6 ± 0.8 (means ± SD) and 2.9 ± 1.5 kPa (P = 0.001), final 11.7 ± 6.4 and 32 ± 15.6 kPa (P matrix fibers. These results suggest that subcutaneous AT has greater capacity for expansion and recovery from mechanical deformation than omental AT.

  11. Comparison of four decontamination treatments on porcine renal decellularized extracellular matrix structure, composition, and support of human renal cortical tubular epithelium cells.

    Science.gov (United States)

    Poornejad, Nafiseh; Nielsen, Jeffery J; Morris, Ryan J; Gassman, Jason R; Reynolds, Paul R; Roeder, Beverly L; Cook, Alonzo D

    2016-03-01

    Engineering whole organs from porcine decellularized extracellular matrix and human cells may lead to a plentiful source of implantable organs. Decontaminating the porcine decellularized extracellular matrix scaffolds is an essential step prior to introducing human cells. However, decontamination of whole porcine kidneys is a major challenge because the decontamination agent or irradiation needs to diffuse deep into the structure to eliminate all microbial contamination while minimizing damage to the structure and composition of the decellularized extracellular matrix. In this study, we compared four decontamination treatments that could be applicable to whole porcine kidneys: 70% ethanol, 0.2% peracetic acid in 1 M NaCl, 0.2% peracetic acid in 4% ethanol, and gamma (γ)-irradiation. Porcine kidneys were decellularized by perfusion of 0.5% (w/v) aqueous solution of sodium dodecyl sulfate and the four decontamination treatments were optimized using segments (n = 60) of renal tissue to ensure a consistent comparison. Although all four methods were successful in decontamination, γ-irradiation was very damaging to collagen fibers and glycosaminoglycans, leading to less proliferation of human renal cortical tubular epithelium cells within the porcine decellularized extracellular matrix. The effectiveness of the other three optimized solution treatments were then all confirmed using whole decellularized porcine kidneys (n = 3). An aqueous solution of 0.2% peracetic acid in 1 M NaCl was determined to be the best method for decontamination of porcine decellularized extracellular matrix.

  12. Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin.

    Science.gov (United States)

    Bae, Jung-Soo; Han, Mira; Shin, Hee Soon; Kim, Min-Kyoung; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-01-04

    Perilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. The present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Human dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. PLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. Our results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Light and electron microscopic study of epithelial cells from the human oviduct and uterus subcultured on extracellular matrix gel.

    Science.gov (United States)

    Eslaminejad, Mohamadreza Baghaban; Valojerdi, Mojtaba Rezazadeh; Ashtiani, Saeed Kazemi; Eftekhari-Yazdi, Poopak

    2007-06-01

    To investigate the structure of epithelial cells from the human oviduct and uterus on extracellular matrix (ECM) gel in the first passage. Human oviducts and endometrial tissues were obtained from patients undergoing total hysterectomy; the epithelial cells, having been isolated by enzyme digestion, were cultured on polystyrene plastic surfaces. The epithelial nature of the cells was confirmed by immunocytochemistry, and their morphology was examined by microscopy. Cells of an epithelial nature were then trypsinized and cultured on an ECM gel-coated filter insert for 5 days. The cells, in parallel with the tissues, were subsequently prepared for transmission electron microscopy. Plastic-cultured cells had no sign of differentiation and appeared as elongated spindle cells in sections. These cells looked columnar and highly polarized after being cultured on ECM gel surfaces. They were similar to epithelial cells from the corresponding tissue fragment. Cultured on ECM gel, the ciliated epithelial cells of human oviducts appeared ultrastructurally similar to glandular cells from the human uterus. Cilia did not form under culture conditions. It seems that human uterine and oviduct epithelial cells can acquire polarized morphology and differentiated states on ECM gel after having lost it on plastic surfaces and that ECM gel by itself is not enough to induce cilia formation in culture.

  14. Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

    Science.gov (United States)

    Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

    2016-02-01

    Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

  15. Skip Regulates TGF-β1-Induced Extracellular Matrix Degrading Proteases Expression in Human PC-3 Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Victor Villar

    2013-01-01

    Full Text Available Purpose. To determine whether Ski-interacting protein (SKIP regulates TGF-β1-stimulated expression of urokinase-type plasminogen activator (uPA, matrix metalloproteinase-9 (MMP-9, and uPA Inhibitor (PAI-1 in the androgen-independent human prostate cancer cell model. Materials and Methods. PC-3 prostate cancer cell line was used. The role of SKIP was evaluated using synthetic small interference RNA (siRNA compounds. The expression of uPA, MMP-9, and PAI-1 was evaluated by zymography assays, RT-PCR, and promoter transactivation analysis. Results. In PC-3 cells TGF-β1 treatment stimulated uPA, PAI-1, and MMP-9 expressions. The knockdown of SKIP in PC-3 cells enhanced the basal level of uPA, and TGF-β1 treatment inhibited uPA production. Both PAI-1 and MMP-9 production levels were increased in response to TGF-β1. The ectopic expression of SKIP inhibited both TGF-β1-induced uPA and MMP-9 promoter transactivation, while PAI-1 promoter response to the factor was unaffected. Conclusions. SKIP regulates the expression of uPA, PAI-1, and MMP-9 stimulated by TGF-β1 in PC-3 cells. Thus, SKIP is implicated in the regulation of extracellular matrix degradation and can therefore be suggested as a novel therapeutic target in prostate cancer treatment.

  16. Human macrophages primed with angiogenic factors show dynamic plasticity, irrespective of extracellular matrix components

    NARCIS (Netherlands)

    Ploeger, Diana T. A.; van Putten, Sander M.; Koerts, Jasper A.; van Luyn, Marja J. A.; Harmsen, Martin C.

    2012-01-01

    Macrophages are important in inflammation as well as in tissue repair processes. They can be activated by various stimuli and classified into two major groups: M1 (classically activated) or M2 (alternatively activated). Inflammation, angiogenesis and matrix remodeling play a major role in tissue rep

  17. Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix.

    Science.gov (United States)

    Gründel, Anne; Jacobs, Enno; Dumke, Roger

    2016-12-01

    Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae.

  18. Extracellular matrix components supporting human islet function in alginate-based immunoprotective microcapsules for treatment of diabetes

    NARCIS (Netherlands)

    Llacua Carrasco, Luis; de Haan, Bart J; Smink, Sandra A; de Vos, Paul

    2016-01-01

    In the pancreas, extracellular matrix (ECM) components play an import role in providing mechanical and physiological support, and also contribute to the function of islets. These ECM-connections are damaged during islet-isolation from the pancreas and are not fully recovered after encapsulation and

  19. Prostaglandins but not leukotrienes alter extracellular matrix protein deposition and cytokine release in primary human airway smooth muscle cells and fibroblasts

    NARCIS (Netherlands)

    Van Ly, David; Burgess, Janette K.; Brock, Thomas G.; Lee, Tak H.; Black, Judith L.; Oliver, Brian G. G.

    2012-01-01

    Van Ly D, Burgess JK, Brock TG, Lee TH, Black JL, Oliver BG. Prostaglandins but not leukotrienes alter extracellular matrix protein deposition and cytokine release in primary human airway smooth muscle cells and fibroblasts. Am J Physiol Lung Cell Mol Physiol 303: L239-L250, 2012. First published Ma

  20. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix.

    Science.gov (United States)

    Ma, Yufei; Ji, Yuan; Huang, Guoyou; Ling, Kai; Zhang, Xiaohui; Xu, Feng

    2015-12-22

    Periodontitis is an inflammatory disease negatively affecting up to 15% of adults worldwide. Periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, where it is necessary to find proper extracellular matrix (ECM) materials (e.g., composition, concentration). In this study, we proposed a bioprinting-based approach to generate nano-liter sized three-dimensional (3D) cell-laden hydrogel array with gradient of ECM components, through controlling the volume ratio of two hydrogels, such as gelatin methacrylate (GelMA) and poly(ethylene glycol) (PEG) dimethacrylate. The resulting cell-laden array with a gradient of GelMA/PEG composition was used to screen human PDLSC response to ECM. The behavior (e.g., cell viability, spreading) of human PDLSCs in GelMA/PEG array were found to be depended on the volume ratios of GelMA/PEG, with cell viability and spreading area decreased along with increasing the ratio of PEG. The developed approach would be useful for screening cell-biomaterial interaction in 3D and promoting regeneration of functional tissue.

  1. Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Jaewoo Pak

    2016-08-01

    Full Text Available This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs and homogenized extracellular matrix (ECM in the form of adipose stromal vascular fraction (SVF, along with hyaluronic acid (HA and platelet-rich plasma (PRP activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI data, functional rating index, range of motion (ROM, and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees.

  2. The generation of hybrid electrospun nanofiber layer with extracellular matrix derived from human pluripotent stem cells, for regenerative medicine applications.

    Science.gov (United States)

    Shtrichman, Ronit; Zeevi-Levin, Naama; Zaid, Rinat; Barak, Efrat; Fishman, Bettina; Ziskind, Anna; Shulman, Rita; Novak, Atara; Avrahami, Ron; Livne, Erella; Lowenstein, Lior; Zussman, Eyal; Itskovitz-Eldor, Joseph

    2014-10-01

    Extracellular matrix (ECM) has been utilized as a biological scaffold for tissue engineering applications in a variety of body systems, due to its bioactivity and biocompatibility. In the current study we developed a modified protocol for the efficient and reproducible derivation of mesenchymal progenitor cells (MPCs) from human embryonic stem cells as well as human induced pluripotent stem cells (hiPSCs) originating from hair follicle keratinocytes (HFKTs). ECM was produced from these MPCs and characterized in comparison to adipose mesenchymal stem cell ECM, demonstrating robust ECM generation by the excised HFKT-iPSC-MPCs. Exploiting the advantages of electrospinning we generated two types of electrospun biodegradable nanofiber layers (NFLs), fabricated from polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA), which provide mechanical support for cell seeding and ECM generation. Elucidating the optimized decellularization treatment we were able to generate an available "off-the-shelf" implantable product (NFL-ECM). Using rat subcutaneous transplantation model we demonstrate that this stem-cell-derived construct is biocompatible and biodegradable and holds great potential for tissue regeneration applications.

  3. Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

    Science.gov (United States)

    Deegan, Daniel B; Zimmerman, Cynthia; Skardal, Aleksander; Atala, Anthony; Shupe, Thomas D

    2015-03-01

    Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture.

  4. Electrical stimulation of neural stem cells mediated by humanized carbon nanotube composite made with extracellular matrix protein.

    Science.gov (United States)

    Kam, Nadine Wong Shi; Jan, Edward; Kotov, Nicholas A

    2009-01-01

    One of the key challenges to engineering neural interfaces is to minimize their immune response toward implanted electrodes. One potential approach is to manufacture materials that bear greater structural resemblance to living tissues and by utilizing neural stem cells. The unique electrical and mechanical properties of carbon nanotubes make them excellent candidates for neural interfaces, but their adoption hinges on finding approaches for "humanizing" their composites. Here we demonstrated the fabrication of layer-by-layer assembled composites from single-walled carbon nanotubes (SWNTs) and laminin, which is an essential part of human extracellular matrix. Laminin-SWNT thin films were found to be conducive to neural stem cells (NSC) differentiation and suitable for their successful excitation. We observed extensive formation of functional neural network as indicated by the presence of synaptic connections. Calcium imaging of the NSCs revealed generation of action potentials upon the application of a lateral current through the SWNT substrate. These results indicate that the protein-SWNT composite can serve as materials foundation of neural electrodes with chemical structure better adapted with long-term integration with the neural tissue.

  5. Role of Age-Associated Alterations of the Dermal Extracellular Matrix Microenvironment in Human Skin Aging

    Science.gov (United States)

    Quan, Taihao; Fisher, Gary J

    2015-01-01

    Human skin is largely composed of a collagen-rich connective tissue, which provides structural and functional support. The collagen-rich connective tissue is produced, organized, and maintained by dermal fibroblasts. During aging, dermal collagen fibrils undergo progressive loss and fragmentation, leading to thin and structurally weakened skin. Age-related alterations of collagen fibrils impairs skin structure and function and creates a tissue microenvironment that promotes age-related skin diseases, such as delayed wound healing and skin cancer development. This review describes cellular mechanisms that give rise to self-perpetuating, collagen fibril fragmentation that creates an age-associated dermal microenvironment (AADM), which contributes to decline of human skin function. PMID:25660807

  6. Role of Age-Associated Alterations of the Dermal Extracellular Matrix Microenvironment in Human Skin Aging

    OpenAIRE

    Quan, Taihao; Fisher, Gary J.

    2015-01-01

    Human skin is largely composed of a collagen-rich connective tissue, which provides structural and functional support. The collagen-rich connective tissue is produced, organized, and maintained by dermal fibroblasts. During aging, dermal collagen fibrils undergo progressive loss and fragmentation, leading to thin and structurally weakened skin. Age-related alterations of collagen fibrils impairs skin structure and function and creates a tissue microenvironment that promotes age-related skin d...

  7. RNA interference targeting extracellular matrix metalloproteinase inducer (CD147) inhibits growth and increases chemosensitivity in human cervical cancer cells.

    Science.gov (United States)

    Zhang, F; Zeng, Y L; Zhang, X G; Chen, W J; Yang, R; Li, S J

    2013-01-01

    Overexpression of extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN CD147) has been implicated in the growth and survival of malignant cells. However, its presence and role in cervical cancer cells has not been well-studied. In the present study, small interfering RNA (siRNA) was designed and synthesized to breakdown the expression of CD147. The present data demonstrated that 24 and 48 hours after transfecting CD147 siRNA, both the CD147 mRNA and protein expression were significantly inhibited as determined by quantitative real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. Meanwhile, simultaneous silencing of CD147 resulted in distinctly increasing MMP-9, VEGF, and MDR-1. Further studies demonstrated decreased CD147 expression, resulted in G1/S phase transition with flow cytometry analysis, as well as the resistance of the cells to 5-FU. These findings provide further evidence that CD147 may become a promising therapeutic target for human cervical cancer and a potential chemotherapy-sensitizing agent.

  8. Inhibition of Histone Deacetylase Activity in Human Endometrial Stromal Cells Promotes Extracellular Matrix Remodelling and Limits Embryo Invasion

    Science.gov (United States)

    Atkinson, Stuart P.; Quiñonero, Alicia; Martínez, Sebastián; Pellicer, Antonio; Simón, Carlos

    2012-01-01

    Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs) to degrade extracellular matrix (ECM) proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs). The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs) that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs) related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA) increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion. PMID:22291969

  9. Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork.

    Directory of Open Access Journals (Sweden)

    Janice A Vranka

    Full Text Available Elevated intraocular pressure (IOP is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm localized throughout the TM layers and Schlemm's canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2 and MMPs (1, 2, 3 were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in

  10. Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

    Science.gov (United States)

    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

  11. Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

    Science.gov (United States)

    Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

    2011-12-01

    The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology.

  12. Culturing on decellularized extracellular matrix enhances antioxidant properties of human umbilical cord-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xiaozhen [School of Engineering, Sun Yat-sen University, Guangzhou 510006 (China); Zhou, Long; Chen, Xi [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Liu, Tao [Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Pan, Guoqing; Cui, Wenguo; Li, Mao; Luo, Zong-Ping [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Pei, Ming [Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV 26506 (United States); Yang, Huilin [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Gong, Yihong, E-mail: gongyih@mail.sysu.edu.cn [School of Engineering, Sun Yat-sen University, Guangzhou 510006 (China); He, Fan, E-mail: fanhe@suda.edu.cn [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China)

    2016-04-01

    Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have attracted great interest in clinical application because of their regenerative potential and their lack of ethical issues. Our previous studies showed that decellularized cell-deposited extracellular matrix (ECM) provided an in vivo-mimicking microenvironment for MSCs and facilitated in vitro cell expansion. This study was conducted to analyze the cellular response of UC-MSCs when culturing on the ECM, including reactive oxygen species (ROS), intracellular antioxidative enzymes, and the resistance to exogenous oxidative stress. After decellularization, the architecture of cell-deposited ECM was characterized as nanofibrous, collagen fibrils and the matrix components were identified as type I and III collagens, fibronectin, and laminin. Compared to tissue culture polystyrene (TCPS) plates, culturing on ECM yielded a 2-fold increase of UC-MSC proliferation and improved the percentage of cells in the S phase by 2.4-fold. The levels of intracellular ROS and hydrogen peroxide (H{sub 2}O{sub 2}) in ECM-cultured cells were reduced by 41.7% and 82.9%, respectively. More importantly, ECM-cultured UC-MSCs showed enhanced expression and activity of intracellular antioxidative enzymes such as superoxide dismutase and catalase, up-regulated expression of silent information regulator type 1, and suppressed phosphorylation of p38 mitogen-activated protein kinase. Furthermore, a continuous treatment with exogenous 100 μM H{sub 2}O{sub 2} dramatically inhibited osteogenic differentiation of UC-MSCs cultured on TCPS, but culturing on ECM retained the differentiation capacity for matrix mineralization and osteoblast-specific marker gene expression. Collectively, by providing sufficient cell amounts and enhancing antioxidant capacity, decellularized ECM can be a promising cell culture platform for in vitro expansion of UC-MSCs. - Highlights: • Decellularization preserved the architecture and components of cell

  13. Fragmentation of extracellular matrix by hypochlorous acid

    DEFF Research Database (Denmark)

    Woods, Alan A; Davies, Michael Jonathan

    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)...

  14. Modulation of extracellular matrix genes reflects the magnitude of physiological adaptation to aerobic exercise training in humans

    Directory of Open Access Journals (Sweden)

    Greenhaff Paul L

    2005-09-01

    Full Text Available Abstract Background Regular exercise reduces cardiovascular and metabolic disease partly through improved aerobic fitness. The determinants of exercise-induced gains in aerobic fitness in humans are not known. We have demonstrated that over 500 genes are activated in response to endurance-exercise training, including modulation of muscle extracellular matrix (ECM genes. Real-time quantitative PCR, which is essential for the characterization of lower abundance genes, was used to examine 15 ECM genes potentially relevant for endurance-exercise adaptation. Twenty-four sedentary male subjects undertook six weeks of high-intensity aerobic cycle training with muscle biopsies being obtained both before and 24 h after training. Subjects were ranked based on improvement in aerobic fitness, and two cohorts were formed (n = 8 per group: the high-responder group (HRG; peak rate of oxygen consumption increased by +0.71 ± 0.1 L min-1; p -1, ns. ECM genes profiled included the angiopoietin 1 and related genes (angiopoietin 2, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1 and 2 (TIE2, vascular endothelial growth factor (VEGF and related receptors (VEGF receptor 1, VEGF receptor 2 and neuropilin-1, thrombospondin-4, α2-macroglobulin and transforming growth factor β2. Results neuropilin-1 (800%; p VEGF receptor 2 (300%; p VEGF receptor 1 mRNA actually declined in the LRG (p TIE1 and TIE2 mRNA levels were unaltered in the LRG, whereas transcription levels of both genes were increased by 2.5-fold in the HRG (p thrombospondin-4 (900%; p α2-macroglobulin (300%, p transforming growth factor β2 transcript increased only in the HRG (330%; p Conclusion We demonstrate for the first time that aerobic training activates angiopoietin 1 and TIE2 genes in human muscle, but only when aerobic capacity adapts to exercise-training. The fourfold-greater increase in aerobic fitness and markedly differing gene expression profile in the HRG indicates that

  15. Cardiac fibroblast-derived extracellular matrix (biomatrix) as a model for the studies of cardiac primitive cell biological properties in normal and pathological adult human heart.

    Science.gov (United States)

    Castaldo, Clotilde; Di Meglio, Franca; Miraglia, Rita; Sacco, Anna Maria; Romano, Veronica; Bancone, Ciro; Della Corte, Alessandro; Montagnani, Stefania; Nurzynska, Daria

    2013-01-01

    Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM) change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease) and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix), composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

  16. Cardiac Fibroblast-Derived Extracellular Matrix (Biomatrix as a Model for the Studies of Cardiac Primitive Cell Biological Properties in Normal and Pathological Adult Human Heart

    Directory of Open Access Journals (Sweden)

    Clotilde Castaldo

    2013-01-01

    Full Text Available Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix, composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

  17. Flax Fiber Hydrophobic Extract Inhibits Human Skin Cells Inflammation and Causes Remodeling of Extracellular Matrix and Wound Closure Activation

    Directory of Open Access Journals (Sweden)

    Monika Styrczewska

    2015-01-01

    Full Text Available Inflammation is the basis of many diseases, with chronic wounds amongst them, limiting cell proliferation and tissue regeneration. Our previous preclinical study of flax fiber applied as a wound dressing and analysis of its components impact on the fibroblast transcriptome suggested flax fiber hydrophobic extract use as an anti-inflammatory and wound healing preparation. The extract contains cannabidiol (CBD, phytosterols, and unsaturated fatty acids, showing great promise in wound healing. In in vitro proliferation and wound closure tests the extract activated cell migration and proliferation. The activity of matrix metalloproteinases in skin cells was increased, suggesting activation of extracellular components remodeling. The expression of cytokines was diminished by the extract in a cannabidiol-dependent manner, but β-sitosterol can act synergistically with CBD in inflammation inhibition. Extracellular matrix related genes were also analyzed, considering their importance in further stages of wound healing. The extract activated skin cell matrix remodeling, but the changes were only partially cannabidiol- and β-sitosterol-dependent. The possible role of fatty acids also present in the extract is suggested. The study shows the hydrophobic flax fiber components as wound healing activators, with anti-inflammatory cannabidiol acting in synergy with sterols, and migration and proliferation promoting agents, some of which still require experimental identification.

  18. Flax Fiber Hydrophobic Extract Inhibits Human Skin Cells Inflammation and Causes Remodeling of Extracellular Matrix and Wound Closure Activation.

    Science.gov (United States)

    Styrczewska, Monika; Kostyn, Anna; Kulma, Anna; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Prescha, Anna; Czuj, Tadeusz; Szopa, Jan

    2015-01-01

    Inflammation is the basis of many diseases, with chronic wounds amongst them, limiting cell proliferation and tissue regeneration. Our previous preclinical study of flax fiber applied as a wound dressing and analysis of its components impact on the fibroblast transcriptome suggested flax fiber hydrophobic extract use as an anti-inflammatory and wound healing preparation. The extract contains cannabidiol (CBD), phytosterols, and unsaturated fatty acids, showing great promise in wound healing. In in vitro proliferation and wound closure tests the extract activated cell migration and proliferation. The activity of matrix metalloproteinases in skin cells was increased, suggesting activation of extracellular components remodeling. The expression of cytokines was diminished by the extract in a cannabidiol-dependent manner, but β-sitosterol can act synergistically with CBD in inflammation inhibition. Extracellular matrix related genes were also analyzed, considering their importance in further stages of wound healing. The extract activated skin cell matrix remodeling, but the changes were only partially cannabidiol- and β-sitosterol-dependent. The possible role of fatty acids also present in the extract is suggested. The study shows the hydrophobic flax fiber components as wound healing activators, with anti-inflammatory cannabidiol acting in synergy with sterols, and migration and proliferation promoting agents, some of which still require experimental identification.

  19. Extracellular matrix component signaling in cancer

    DEFF Research Database (Denmark)

    Multhaupt, Hinke A. B.; Leitinger, Birgit; Gullberg, Donald

    2016-01-01

    Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization and motil...... as well as matrix constitution and protein crosslinking. Here we summarize roles of the three major matrix receptor types, with emphasis on how they function in tumor progression. [on SciFinder(R)]......Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization...... and motility but also provides survival and proliferation cues. The major classes of cell surface receptors for matrix macromols. are the integrins, discoidin domain receptors, and transmembrane proteoglycans such as syndecans and CD44. Cells respond not only to specific ligands, such as collagen, fibronectin...

  20. Differential expression of extracellular matrix proteins in senescent and young human fibroblasts: a comparative proteomics and microarray study.

    Science.gov (United States)

    Yang, Kyeong Eun; Kwon, Joseph; Rhim, Ji-Heon; Choi, Jong Soon; Kim, Seung Il; Lee, Seung-Hoon; Park, Junsoo; Jang, Ik-Soon

    2011-07-01

    The extracellular matrix (ECM) provides an essential structural framework for cell attachment, proliferation, and differentiation, and undergoes progressive changes during senescence. To investigate changes in protein expression in the extracellular matrix between young and senescent fibroblasts, we compared proteomic data (LTQ-FT) with cDNA microarray results. The peptide counts from the proteomics analysis were used to evaluate the level of ECM protein expression by young cells and senescent cells, and ECM protein expression data were compared with the microarray data. After completing the comparative analysis, we grouped the genes into four categories. Class I included genes with increased expression levels in both analyses, while class IV contained genes with reduced expression in both analyses. Class II and Class III contained genes with an inconsistent expression pattern. Finally, we validated the comparative analysis results by examining the expression level of the specific gene from each category using Western blot analysis and semiquantitative RT-PCR. Our results demonstrate that comparative analysis can be used to identify differentially expressed genes.

  1. Ability of sodium copper chlorophyllin complex to repair photoaged skin by stimulation of biomarkers in human extracellular matrix

    Directory of Open Access Journals (Sweden)

    McCook JP

    2016-07-01

    Full Text Available John P McCook,1 Thomas J Stephens,2 Lily I Jiang,2 Robert M Law,3 Vincent Gotz4 1Discovery Partners LLC, Frisco, 2Thomas J. Stephens & Associates, Inc., Richardson, 3ProPath, Dallas, TX, 4MDRejuvena, Inc., San Diego, CA, USA Purpose: To examine the effect of sodium copper chlorophyllin complex on the expression of biomarkers of photoaged dermal extracellular matrix indicative of skin repair.Patients and methods: Following a previously published 12-day clinical assessment model, skin biopsy samples from the forearms of four healthy females with signs of photoaged skin were obtained and samples were analyzed by immunohistochemistry for key biomarkers of aging skin after each subject was treated with a test material consisting of a gel containing a liposomal dispersion of sodium copper chlorophyllin complex 0.05%, a positive control of tretinoin cream 0.025%, and an untreated negative control.Results: There was a statistically significantly greater amount of fibrillin/amyloid P and epidermal mucins found for skin treated with the test material containing 0.05% sodium copper chlorophyllin complex and the reference control tretinoin 0.025% cream compared to the negative control (untreated site. Expression of procollagen 1 and dermal mucin also showed a greater presence in the samples treated with the test material and the reference control compared to the negative control, though the differences were not statistically significant. No adverse events were observed or reported by the subjects during the course of the study.Conclusion: The results of this human biopsy study suggest that both retinoids and sodium copper chlorophyllin complex have beneficial effects on biomarkers of photoaged skin. Products containing both sodium copper chlorophyllin complex and retinols may provide a dual approach to reversing age-related decreases in hyaluronic acid (HA in the skin: inhibition of the breakdown of HA via sodium copper chlorophyllin complex by inhibition

  2. Extracellular matrix and tissue engineering applications

    NARCIS (Netherlands)

    Fernandes, Hugo; Moroni, Lorenzo; Blitterswijk, van Clemens; Boer, de Jan

    2009-01-01

    The extracellular matrix is a key component during regeneration and maintenance of tissues and organs, and it therefore plays a critical role in successful tissue engineering as well. Tissue engineers should recognise that engineering technology can be deduced from natural repair processes. Due to a

  3. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Kevin Dzobo

    2016-08-01

    Full Text Available Mesenchymal stromal/stem cells (MSCs represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4, SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

  4. Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis.

    Science.gov (United States)

    Oostendorp, Corien; Meyer, Sarah; Sobrio, Monia; van Arendonk, Joyce; Reichmann, Ernst; Daamen, Willeke F; van Kuppevelt, Toin H

    2017-05-01

    Treatment of full-thickness skin defects with split-thickness skin grafts is generally associated with contraction and scar formation and cellular skin substitutes have been developed to improve skin regeneration. The evaluation of cultured skin substitutes is generally based on qualitative parameters focusing on histology. In this study we focused on quantitative evaluation to provide a template for comparison of human bio-engineered skin substitutes between clinical and/or research centers, and to supplement histological data. We focused on extracellular matrix proteins since these components play an important role in skin regeneration. As a model we analyzed the human dermal substitute denovoDerm and the dermo-epidermal skin substitute denovoSkin. The quantification of the extracellular matrix proteins type III collagen and laminin 5 in tissue homogenates using western blotting analysis and ELISA was not successful. The same was true for assaying lysyl oxidase, an enzyme involved in crosslinking of matrix molecules. As an alternative, gene expression levels were measured using qPCR. Various RNA isolation procedures were probed. The gene expression profile for specific dermal and epidermal genes could be measured reliably and reproducibly. Differences caused by changes in the cell culture conditions could easily be detected. The number of cells in the skin substitutes was measured using the PicoGreen dsDNA assay, which was found highly quantitative and reproducible. The (dis) advantages of assays used for quantitative evaluation of skin substitutes are discussed.

  5. Matrix Extracellular Phosphoglycoprotein Inhibits Phosphate Transport

    OpenAIRE

    Marks, J; Churchill, L J; Debnam, E. S.; Unwin, R J

    2008-01-01

    The role of putative humoral factors, known as phosphatonins, in phosphate homeostasis and the relationship between phosphate handling by the kidney and gastrointestinal tract are incompletely understood. Matrix extracellular phosphoglycoprotein (MEPE), one of several candidate phosphatonins, promotes phosphaturia, but whether it also affects intestinal phosphate absorption is unknown. Here, using the in situ intestinal loop technique, we demonstrated that short-term infusion of MEPE inhibits...

  6. Engineering hydrogels as extracellular matrix mimics

    OpenAIRE

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

    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....

  7. Involvement of extracellular matrix constituents in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Extracellular matrix proteins involved in pseudoislets formation.

    Science.gov (United States)

    Maillard, Elisa; Sencier, Marie-Christine; Langlois, A; Bietiger, William; Krafft, Mp; Pinget, Michel; Sigrist, Séverine

    2009-01-01

    Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the β-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and β1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral β-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of β1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.

  9. Expression of extracellular matrix components and related growth factors in human tendon and muscle after acute exercise

    DEFF Research Database (Denmark)

    Heinemeier, K M; Bjerrum, S S; Schjerling, P

    2013-01-01

    the patellar tendon and vastus lateralis muscle of each leg at 2 (n = 10), 6 (n = 11), or 26 h (n = 10) after exercise. Levels of messenger ribonucleic acid mRNA for collagens, noncollagenous matrix proteins, and growth factors were measured with real-time reverse transcription polymerase chain reaction......Acute kicking exercise induces collagen synthesis in both tendon and muscle in humans, but it is not known if this relates to increased collagen transcription and if other matrix genes are regulated. Young men performed 1 h of one-leg kicking at 67% of max workload. Biopsies were taken from....... In tendon, gene expression was unchanged except for a decrease in insulin-like growth factor-IEa (IGF-IEa; P ...

  10. Balance between apoptosis or survival induced by changes in extracellular-matrix composition in human mesangial cells: a key role for ILK-NFκB pathway.

    Science.gov (United States)

    del Nogal, María; Luengo, Alicia; Olmos, Gemma; Lasa, Marina; Rodriguez-Puyol, Diego; Rodriguez-Puyol, Manuel; Calleros, Laura

    2012-12-01

    Renal fibrosis is the final outcome of many clinical conditions that lead to chronic renal failure, characterized by a progressive substitution of cellular elements by extracellular-matrix proteins, in particular collagen type I. The aim of this study was to identify the mechanisms responsible for human mesangial cell survival, conditioned by changes in extracellular-matrix composition. Our results indicate that collagen I induces apoptosis in cells but only after inactivation of the pro-survival factor NFκB by either the super-repressor IκBα or the PDTC inhibitor. Collagen I activates a death pathway, through ILK/GSK-3β-dependent Bim expression. Moreover, collagen I significantly increases NFκB-dependent transcription, IκBα degradation and p65/NFκB translocation to the nucleus; it activates β1 integrin and this is accompanied by increased activity of ILK which leads to AKT activation. Knockdown of ILK or AKT with small interfering RNA suppresses the increase in NFκB activity. NFκB mediates cell survival through the antiapoptotic protein Bcl-xL. Our data suggest that human mesangial cells exposed to abnormal collagen I are protected against apoptosis by a complex mechanism involving integrin β1/ILK/AKT-dependent NFκB activation with consequent Bcl-xL overexpression, that opposes a simultaneously activated ILK/GSK-3β-dependent Bim expression and this dual mechanism may play a role in the progression of glomerular dysfunction.

  11. Lung extracellular matrix and redox regulation.

    Science.gov (United States)

    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

  12. Lung extracellular matrix and redox regulation

    Directory of Open Access Journals (Sweden)

    Walter H. Watson

    2016-08-01

    Full Text Available 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

  13. Thermoreversible copolymer gels for extracellular matrix.

    Science.gov (United States)

    Vernon, B; Kim, S W; Bae, Y H

    2000-07-01

    To improve the properties of a reversible synthetic extracellular matrix based on a thermally reversible polymer, copolymers of N-isopropylacrylamide and acrylic acid were prepared in benzene with varying contents of acrylic acid (0 to 3%) and the thermal properties were evaluated. The poly(N-isopropylacrylamide) and copolymers made with acrylic acid had molecular weights from 0.8 to 1.7 x10(6) D. Differential scanning calorimetry (DSC) showed the high-molecular-weight acrylic acid copolymers had similar onset temperatures to the homopolymers, but the peak width was considerably increased with increasing acrylic acid content. DSC and cloud point measurements showed that polymers with 0 to 3% acrylic acid exhibit a lower critical solution temperature (LCST) transition between 30 degrees and 37 degrees C. In swelling studies, the homopolymer showed significant syneresis at temperatures above 31 degrees C. Copolymers with 1 and 1.5% showed syneresis beginning at 32 degrees and 37 degrees C, respectively. At 37 degrees C the copolymers with 1.5-3% acrylic acid showed little or no syneresis. Due to the high water content and a transition near physiologic conditions (below 37 degrees C), the polymers with 1.5-2.0% acrylic acid exhibited properties that would be useful in the development of a refillable synthetic extracellular matrix. Such a matrix could be applied to several cell types, including islets of Langerhans, for a biohybrid artificial pancreas.

  14. Flavonoids suppress human glioblastoma cell growth by inhibiting cell metabolism, migration, and by regulating extracellular matrix proteins and metalloproteinases expression.

    Science.gov (United States)

    Santos, Balbino L; Oliveira, Mona N; Coelho, Paulo L C; Pitanga, Bruno P S; da Silva, Alessandra B; Adelita, Taís; Silva, Victor Diógenes A; Costa, Maria de F D; El-Bachá, Ramon S; Tardy, Marcienne; Chneiweiss, Hervé; Junier, Marie-Pierre; Moura-Neto, Vivaldo; Costa, Silvia L

    2015-12-01

    The malignant gliomas are very common primary brain tumors with poor prognosis, which require more effective therapies than the current used, such as with chemotherapy drugs. In this work, we investigated the effects of several polyhydroxylated flavonoids namely, rutin, quercetin (F7), apigenin (F32), chrysin (F11), kaempferol (F12), and 3',4'-dihydroxyflavone (F2) in human GL-15 glioblastoma cells. We observed that all flavonoids decreased the number of viable cells and the mitochondrial metabolism. Furthermore, they damaged mitochondria and rough endoplasmic reticulum, inducing apoptosis. Flavonoids also induced a delay in cell migration, related to a reduction in filopodia-like structures on the cell surface, reduction on metalloproteinase (MMP-2) expression and activity, as well as an increase in intra- and extracellular expression of fibronectin, and intracellular expression of laminin. Morphological changes were also evident in adherent cells characterized by the presence of a condensed cell body with thin and long cellular processes, expressing glial fibrillary acidic protein (GFAP). Therefore, these flavonoids should be tested as potential antitumor agents in vitro and in vivo in other malignant glioma models.

  15. Bidirectional extracellular matrix signaling during tissue morphogenesis

    Science.gov (United States)

    Gjorevski, Nikolce; Nelson, Celeste M.

    2009-01-01

    Normal tissue development and function are regulated by the interplay between cells and their surrounding extracellular matrix (ECM). The ECM provides biochemical and mechanical contextual information that is conveyed from the cell membrane through the cytoskeleton to the nucleus to direct cell phenotype. Cells, in turn, remodel the ECM and thereby sculpt their local microenvironment. Here we review the mechanisms by which cells interact with, respond to, and influence the ECM, with particular emphasis placed on the role of this bidirectional communication during tissue morphogenesis. We also discuss the implications for successful engineering of functional tissues ex vivo. PMID:19896886

  16. Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

    Directory of Open Access Journals (Sweden)

    M. Paez-Pereda

    2005-10-01

    Full Text Available The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

  17. Extracellular Matrix Modulates Morphology, Growth, Oxidative Stress Response and Functionality of Human Skin Fibroblasts during Aging In Vitro

    DEFF Research Database (Denmark)

    Jørgensen, Peter; Rattan, Suresh

    2014-01-01

    The Hayflick system of cellular aging and replicative senescence in vitro has been used widely in both basic and applied research in biogerontology. The state of replicative senescence is generally considered to be irreversible, but is modifiable by genetic and environmental manipulations. Some...... recent observations indicate that replicative lifespan, senescence and functionality of cells in vitro can be significantly affected by the quality of the extra cellular matrix (ECM). Following up on those reports, here we show that using the ECM prepared from early passage young cells, partial...... rejuvenation of serially passaged human facial skin fibroblasts was possible in pre-senescent middle-aged cells, but not in fully senescent late passage cells. ECM from young cells improved the appearance, viability, stress tolerance and wound healing ability of skin fibroblasts. Furthermore, young ECM...

  18. Resorbable extracellular matrix grafts in urologic reconstruction

    Directory of Open Access Journals (Sweden)

    Richard A. Santucci

    2005-06-01

    Full Text Available PURPOSE: There is an increasingly large body of literature concerning tissue-engineering products that may be used in urology. Some of these are quite complex (such as multilayer patient-specific cell-seeded implants yet the most simple and successful products to date are also the most uncomplicated: resorbable acellular extra-cellular matrices (ECMs harvested from animals. ECMs have been used in a variety of difficult urologic reconstruction problems, and this review is intended to summarize this complex literature for the practicing urologist. METHODS: Medline search of related terms such as "SIS, small intestinal submucosa, ECM, extracellular matrix, acellular matrix and urologic reconstruction". Manuscripts missed in the initial search were taken from the bibliographies of the primary references. RESULTS: Full review of potential clinical uses of resorbable extra-cellular matrices in urologic reconstruction. CONCLUSIONS: Currently, the "state of the art" in tissue engineering solutions for urologic reconstruction means resorbable acellular xenograft matrices. They show promise when used as a pubovaginal sling or extra bolstering layers in ureteral or urethral repairs, although recent problems with inflammation following 8-ply pubovaginal sling use and failures after 1- and 4-ply SIS repair of Peyronie's disease underscore the need for research before wide adoption. Preliminary data is mixed concerning the potential for ECM urethral patch graft, and more data is needed before extended uses such as bladder augmentation and ureteral replacement are contemplated. The distant future of ECMs in urology likely will include cell-seeded grafts with the eventual hope of producing "off the shelf" replacement materials. Until that day arrives, ECMs only fulfill some of the requirements for the reconstructive urologist.

  19. Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions.

    Directory of Open Access Journals (Sweden)

    Thomas J Kean

    Full Text Available Current tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5% oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential.Porcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%, and atmospheric (20% oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically.Human chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions

  20. Hypoxia-increased expression of genes involved in inflammation, dedifferentiation, pro-fibrosis, and extracellular matrix remodeling of human bladder smooth muscle cells.

    Science.gov (United States)

    Wiafe, Bridget; Adesida, Adetola; Churchill, Thomas; Adewuyi, Esther Ekpe; Li, Zack; Metcalfe, Peter

    2017-01-01

    Partial bladder outlet obstruction (pBOO) is characterized by exaggerated stretch, hydrodynamic pressure, and inflammation which cause significant damage and fibrosis to the bladder wall. Several studies have implicated hypoxia in its pathophysiology. However, the isolated progressive effects of hypoxia on bladder cells are not yet defined. Sub-confluent normal human bladder smooth muscle cells (hbSMC) were cultured in 3% O2 tension for 2, 24, 48, and 72 h. RNA, cellular proteins, and secreted proteins were used for gene expression analysis, immunoblotting, and ELISA, respectively. Transcription of hypoxia-inducible factor (HIF)1α and HIF2α were transiently induced after 2 h of hypoxia (p inflammation, de-differentiation, pro-fibrotic changes, and increased extracellular matrix expression. This elucidates mechanisms of hypoxia-driven bladder deterioration in bladder cells, which is important in tailoring in vivo experiments and may ultimately translate into improved clinical outcomes.

  1. 人脂肪组织细胞外基质支架的构建%Construction of scaffold with human extracellular matrix from adipose tissue

    Institute of Scientific and Technical Information of China (English)

    察鹏飞; 高建华; 陈阳; 鲁峰

    2012-01-01

    目的 探讨从人脂肪组织中提取细胞外基质并构建支架的方法,研究其结构特点,分析其作为脂肪组织工程支架的可行性.方法 收集抽脂术患者的脂肪组织,共7例,每例约310 ml,10 ml用来分离培养脂肪来源干细胞,300 ml提取细胞外基质,并制备成粉末状,扫描电镜观察其表面结构.DiI荧光标记脂肪来源干细胞,统计荧光标记后细胞存活率;将荧光标记前、后的细胞与支架粘附,检测其粘附率,所得数据用SPSS 13.0软件两样本t检验进行统计学比较,分析标记前后细胞粘附率有无差异;荧光显微镜下观察细胞在支架表面生长情况.结果 从人脂肪组织中提取得到脂肪来源干细胞和细胞外基质粉末.脂肪来源干细胞具有成脂、成软骨和成骨分化的能力;扫描电镜观察细胞外基质粉末具有多孔、粗糙表面和光滑表面的结构.脂肪来源干细胞与支架粘附较好;DiI标记前、后细胞粘附率分别为(88.81±4.81)%和(86.48±4.58)%,两样本t检验,P=0.371,差异无统计学意义(P>0.05).荧光显微镜下脂肪来源干细胞在细胞外基质支架上生长状态良好.结论 人脂肪组织细胞外基质粉末容易获取,形状和颗粒体积具有高度的多样性,表面积较大,有利于脂肪干细胞的粘附和增殖,可作为一种较理想的脂肪组织工程支架材料.%Objective To investigate the feasibility of constructing scaffold for tissue engineering with human extracellular matrix from adipose tissue.Methods Fresh human adipose tissue was obtained by liposuction in 7 women who undergone liposuction.One part of the fat was used to isolate the adiposederived stem cells(ADSCs),the other part was used to extract human extracellular matrix powder.After removing blood and oil components,the tissue was homogenized,centrifuged,freeze-dried,and crushed to powder by instrument.The structure of human ECM powder was observed with electron microscopy. The

  2. The extracellular matrix in breast cancer.

    Science.gov (United States)

    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.

  3. Extracellular Matrix Molecules Facilitating Vascular Biointegration

    Directory of Open Access Journals (Sweden)

    Martin K.C. Ng

    2012-08-01

    Full Text Available All vascular implants, including stents, heart valves and graft materials exhibit suboptimal biocompatibility that significantly reduces their clinical efficacy. A range of biomolecules in the subendothelial space have been shown to play critical roles in local regulation of thrombosis, endothelial growth and smooth muscle cell proliferation, making these attractive candidates for modulation of vascular device biointegration. However, classically used biomaterial coatings, such as fibronectin and laminin, modulate only one of these components; enhancing endothelial cell attachment, but also activating platelets and triggering thrombosis. This review examines a subset of extracellular matrix molecules that have demonstrated multi-faceted vascular compatibility and accordingly are promising candidates to improve the biointegration of vascular biomaterials.

  4. Defining the extracellular matrix using proteomics

    Science.gov (United States)

    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

  5. Starved epithelial cells uptake extracellular matrix for survival

    Science.gov (United States)

    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

  6. Distribution of cytoskeletal proteins, integrins, leukocyte adhesion molecules and extracellular matrix proteins in plastic-embedded human and rat kidneys

    NARCIS (Netherlands)

    van Goor, H; Coers, W; van der Horst, MLC; Suurmeijer, AJH

    2001-01-01

    OBJECTIVE: To study the distribution of cytoskeletal proteins (actin, alpha -actinin, vinculin, beta -tubulin, keratin, vimentin, desmin), adhesion molecules for cell-matrix interations (very later antigens [VLA1-6], beta1, beta2 [CD18], vitronectin receptor [alphav beta3], CD 11b), leukocyte adhesi

  7. Transplantation of three-dimensional artificial human vascular tissues fabricated using an extracellular matrix nanofilm-based cell-accumulation technique.

    Science.gov (United States)

    Asano, Yoshiya; Shimoda, Hiroshi; Okano, Daisuke; Matsusaki, Michiya; Akashi, Mitsuru

    2017-04-01

    We have established a novel three-dimensional (3D) tissue-constructing technique, referred to as the 'cell-accumulation method', which is based on the self-assembly of cultured human cells. In this technique, cells are coated with fibronectin and gelatin to construct extracellular matrix (ECM) nanofilms and cultured to form multi-layers in vitro. By using this method, we have successfully fabricated artificial tissues with vascular networks constructed by co-cultivation of human umbilical vein-derived vascular endothelial cells between multi-layers of normal human dermal fibroblasts. In this study, to assess these engineered vascular tissues as therapeutic implants, we transplanted the 3D human tissues with microvascular networks, fabricated based on the cell-accumulation method, onto the back skin of nude mice. After the transplantation, we found vascular networks with perfusion of blood in the transplanted graft. At the boundary between host and implanted tissue, connectivity between murine and human vessels was found. Transmission electron microscopy of the implanted artificial vascular tubules demonstrated the ultrastructural features of blood capillaries. Moreover, maturation of the vascular tissues after transplantation was shown by the presence of pericyte-like cells and abundant collagen fibrils in the ECM surrounding the vasculature. These results demonstrated that artificial human vascular tissues constructed by our method were engrafted and matured in animal skin. In addition, the implanted artificial human vascular networks were connected with the host circulatory system by anastomosis. This method is an attractive technique for engineering prevascularized artificial tissues for transplantation. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium

    National Research Council Canada - National Science Library

    Jabaji, Ziyad; Brinkley, Garrett J; Khalil, Hassan A; Sears, Connie M; Lei, Nan Ye; Lewis, Michael; Stelzner, Matthias; Martín, Martín G; Dunn, James C Y

    2014-01-01

    .... There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells...

  9. Type I Collagen as an Extracellular Matrix for the In Vitro Growth of Human Small Intestinal Epithelium: e107814

    National Research Council Canada - National Science Library

    Ziyad Jabaji; Garrett J Brinkley; Hassan A Khalil; Connie M Sears; Nan Ye Lei; Michael Lewis; Matthias Stelzner; Martín G Martín; James C Y Dunn

    2014-01-01

    .... There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells...

  10. Sequenced response of extracellular matrix deadhesion and fibrotic regulators after muscle damage is involved in protection against future injury in human skeletal muscle.

    Science.gov (United States)

    Mackey, Abigail L; Brandstetter, Simon; Schjerling, Peter; Bojsen-Moller, Jens; Qvortrup, Klaus; Pedersen, Mette M; Doessing, Simon; Kjaer, Michael; Magnusson, S Peter; Langberg, Henning

    2011-06-01

    The purpose of this study was to test the hypothesis that remodeling of skeletal muscle extracellular matrix (ECM) is involved in protecting human muscle against injury. Biopsies were obtained from medial gastrocnemius muscles after a single bout of electrical stimulation (B) or a repeated bout (RB) 30 d later, or 30 d after a single stimulation bout (RBc). A muscle biopsy was collected from the control leg for comparison with the stimulated leg. Satellite cell content, tenascin C, and muscle regeneration were assessed by immunohistochemistry; real-time PCR was used to measure mRNA levels of collagens, laminins, heat-shock proteins (HSPs), inflammation, and related growth factors. The large responses of HSPs, CCL2, and tenascin C detected 48 h after a single bout were attenuated in the RB trial, indicative of protection against injury. Satellite cell content and 12 target genes, including IGF-1, were elevated 30 d after a single bout. Among those displaying the greatest difference vs. control muscle, ECM laminin-β1 and collagen types I and III were elevated ∼6- to 9-fold (P<0.001). The findings indicate that the sequenced events of load-induced early deadhesion and later strengthening of skeletal muscle ECM play a role in protecting human muscle against future injury.

  11. Reduced Expression of Cytoskeletal and Extracellular Matrix Genes in Human Adult Retinal Pigment Epithelium Cells Exposed to Simulated Microgravity

    DEFF Research Database (Denmark)

    Corydon, Thomas J; Mann, Vivek; Slumstrup, Lasse;

    2016-01-01

    BACKGROUND/AIMS: Microgravity (µg) has adverse effects on the eye of humans in space. The risk of visual impairment is therefore one of the leading health concerns for NASA. The impact of µg on human adult retinal epithelium (ARPE-19) cells is unknown. METHODS: In this study we investigated the i...

  12. Engineering hydrogels as extracellular matrix mimics.

    Science.gov (United States)

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

    2010-04-01

    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.

  13. Micro- and macrorheology of jellyfish extracellular matrix.

    Science.gov (United States)

    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.

  14. Force spectroscopy of hepatocytic extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Yongsunthon, R., E-mail: YongsuntR@Corning.com [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States); Baker, W.A.; Bryhan, M.D.; Baker, D.E.; Chang, T.; Petzold, O.N.; Walczak, W.J.; Liu, J.; Faris, R.A.; Senaratne, W.; Seeley, L.A.; Youngman, R.E. [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States)

    2009-07-15

    We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular 'footprints' indicated that the cells secrete large polymers (e.g., 3.5 {mu}m contour length and estimated MW 1000 kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.

  15. Extracellular matrix components in peripheral nerve regeneration.

    Science.gov (United States)

    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.

  16. Extracellular matrix gel is necessary for in vitro cultivation of insulin producing cells from human umbilical cord blood derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    GAO Feng; WU De-quan; HU Yan-hua; JIN Guang-xin

    2008-01-01

    Background Pancreatic islet cell transplantation is an effective approach to treat type 1 diabetes. However, this therapy is not widely used because of the severe shortage of transplantable donor islets. This study investigated whether mesenchymal stem cells (MSCs) derived from human umbilical cord blood (UCB) could be transdifferentiated into insulin producing cells in vitro and the role of extracellular matrix (ECM) gel in this procedure.Methods Human UCB samples were collected and MSCs were isolated. MSCs specific marker proteins were analyzed by a flow cytometer. The capacities of osteoblast and adipocyte to differentiate were tested. Differentiation into islet like cell was induced by a 15-day protocol with or without ECM gel. Pancreatic characteristics were evaluated with immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Insulin content and release in response to glucose stimulation were detected with chemiluminescent immunoassay system.Results Sixteen MSCs were isolated from 42 term human UCB units (38%). Human UCB-MSCs expressed MSCs specific markers and could be induced in vitro into osteoblast and adipocyte. Islet like cell clusters appeared about 9 days after pancreatic differentiation in the inducing system with ECM gel. The insulin positive cells accounted for (25.2±3.4)% of the induced cells. The induced cells expressed islet related genes and hormones, but were not very responsive to glucose challenge. When MSCs were induced without ECM gel, clusters formation and secretion of functional islet proteins could not be observed.Conclusions Human UCB-MSCs can differentiate into islet like cells in vitro and ECM gel plays an important role in pancreatic endocrine cell maturation and formation of three dimensional structures.

  17. Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium.

    Directory of Open Access Journals (Sweden)

    Ziyad Jabaji

    Full Text Available We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells.Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry.Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages.Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet

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

    Directory of Open Access Journals (Sweden)

    Wendell Q Sun

    2013-09-01

    Full Text Available 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

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

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    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.

  20. Role of Age-Associated Alterations of the Dermal Extracellular Matrix Microenvironment in Human Skin Aging: A Mini-Review.

    Science.gov (United States)

    Quan, Taihao; Fisher, Gary J

    2015-01-01

    Human skin is largely composed of a collagen-rich connective tissue, which provides structural and functional support. The collagen-rich connective tissue is produced, organized, and maintained by dermal fibroblasts. During aging, dermal collagen fibrils undergo progressive loss and fragmentation, leading to thin and structurally weakened skin. Age-related alterations of collagen fibrils impairs skin structure and function and creates a tissue microenvironment that promotes age-related skin diseases, such as delayed wound healing and skin cancer development. This mini-review describes cellular mechanisms that give rise to self-perpetuating, collagen fibril fragmentation that creates an age-associated dermal microenvironment, which contributes to decline of human skin function.

  1. Comparison of osteoclastogenesis and resorption activity of human osteoclasts on tissue culture polystyrene and on natural extracellular bone matrix in 2D and 3D.

    Science.gov (United States)

    Kleinhans, C; Schmid, F F; Schmid, F V; Kluger, P J

    2015-07-10

    Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (αVβ3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts.

  2. Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating.

    Science.gov (United States)

    Sorkio, Anni; Hongisto, Heidi; Kaarniranta, Kai; Uusitalo, Hannu; Juuti-Uusitalo, Kati; Skottman, Heli

    2014-02-01

    Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

  3. Reactivating the extracellular matrix synthesis of sulfated glycosaminoglycans and proteoglycans to improve the human skin aspect and its mechanical properties

    Directory of Open Access Journals (Sweden)

    Chajra H

    2016-12-01

    Full Text Available Hanane Chajra,1 Daniel Auriol,1 Francine Joly,2 Aurélie Pagnon,3 Magda Rodrigues,4 Sophie Allart,4 Gérard Redziniak,5 Fabrice Lefevre1 1Libragen, Induchem (Givaudan Active Beauty, Toulouse, 2Sephra Pharma, Puteaux, 3Novotec, Bron, 4Centre de Physiopathologie de Toulouse-Purpan, Toulouse, 5Cosmetic Inventions, Antony, France Background: The aim of this study was to demonstrate that a defined cosmetic composition is able to induce an increase in the production of sulfated glycosaminoglycans (sGAGs and/or proteoglycans and finally to demonstrate that the composition, through its combined action of enzyme production and synthesis of macromolecules, modulates organization and skin surface aspect with a benefit in antiaging applications. Materials and methods: Gene expression was studied by quantitative reverse transcription polymerase chain reaction using normal human dermal fibroblasts isolated from a 45-year-old donor skin dermis. De novo synthesis of sGAGs and proteoglycans was determined using Blyscan™ assay and/or immunohistochemical techniques. These studies were performed on normal human dermal fibroblasts (41- and 62-year-old donors and on human skin explants. Dermis organization was studied either ex vivo on skin explants using bi-photon microscopy and transmission electron microscopy or directly in vivo on human volunteers by ultrasound technique. Skin surface modification was investigated in vivo using silicone replicas coupled with macrophotography, and the mechanical properties of the skin were studied using Cutometer. Results: It was first shown that mRNA expression of several genes involved in the synthesis pathway of sGAG was stimulated. An increase in the de novo synthesis of sGAGs was shown at the cellular level despite the age of cells, and this phenomenon was clearly related to the previously observed stimulation of mRNA expression of genes. An increase in the expression of the corresponding core protein of decorin, perlecan

  4. Reduced Expression of Cytoskeletal and Extracellular Matrix Genes in Human Adult Retinal Pigment Epithelium Cells Exposed to Simulated Microgravity

    Directory of Open Access Journals (Sweden)

    Thomas J. Corydon

    2016-11-01

    Full Text Available Background/Aims: Microgravity (µg has adverse effects on the eye of humans in space. The risk of visual impairment is therefore one of the leading health concerns for NASA. The impact of µg on human adult retinal epithelium (ARPE-19 cells is unknown. Methods: In this study we investigated the influence of simulated µg (s-µg; 5 and 10 days (d, using a Random Positioning Machine (RPM, on ARPE-19 cells. We performed phase-contrast/fluorescent microscopy, qRT-PCR, Western blotting and pathway analysis. Results: Following RPM-exposure a subset of ARPE-19 cells formed multicellular spheroids (MCS, whereas the majority of the cells remained adherent (AD. After 5d, alterations of F-actin and fibronectin were observed which reverted after 10d-exposure, suggesting a time-dependent adaptation to s-µg. Gene expression analysis of 12 genes involved in cell structure, shape, adhesion, migration, and angiogenesis suggested significant changes after a 10d-RPM-exposure. 11 genes were down-regulated in AD and MCS 10d-RPM-samples compared to 1g, whereas FLK1 was up-regulated in 5d- and 10d-RPM-MCS-samples. Similarly, TIMP1 was up-regulated in 5d-RPM-samples, whereas the remaining genes were down-regulated in 5d-RPM-samples. Western blotting revealed similar changes in VEGF, β-actin, laminin and fibronectin of 5d-RPM-samples compared to 10d, whereas different alterations of β-tubulin and vimentin were observed. The pathway analysis showed complementing effects of VEGF and integrin β-1. Conclusions: These findings clearly show that s-µg induces significant alterations in the F-actin-cytoskeleton and cytoskeleton-related proteins of ARPE-19, in addition to changes in cell growth behavior and gene expression patterns involved in cell structure, growth, shape, migration, adhesion and angiogenesis.

  5. Micropatterning Extracellular Matrix Proteins on Electrospun Fibrous Substrate Promote Human Mesenchymal Stem Cell Differentiation Toward Neurogenic Lineage.

    Science.gov (United States)

    Li, Huaqiong; Wen, Feng; Chen, Huizhi; Pal, Mintu; Lai, Yuekun; Zhao, Allan Zijian; Tan, Lay Poh

    2016-01-13

    In this study, hybrid micropatterned grafts constructed via a combination of microcontact printing and electrospinning techniques process were utilized to investigate the influencing of patterning directions on human mesenchymal stem cells (hMSCs) differentiation to desired phenotypes. We found that the stem cells could align and elongate along the direction of the micropattern, where they randomly distributed on nonmicropatterned surfaces. Concomitant with patterning effect of component on stem cell alignment, a commensurate increase on the expression of neural lineage commitment markers, such as microtubule associated protein 2 (MAP2), Nestin, NeuroD1, and Class III β-Tubulin, were revealed from mRNA expression by quantitative Real Time PCR (qRT-PCR) and MAP2 expression by immunostaining. In addition, the effect of electrospun fiber orientation on cell behaviors was further examined. An angle of 45° between the direction of micropatterning and orientation of aligned fibers was verified to greatly prompt the outgrowth of filopodia and neurogenesis of hMSCs. This study demonstrates that the significance of hybrid components and electrospun fiber alignment in modulating cellular behavior and neurogenic lineage commitment of hMSCs, suggesting promising application of porous scaffolds with smart component and topography engineering in clinical regenerative medicine.

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

    Science.gov (United States)

    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.

  7. Effects of ionizing radiation on extracellular matrix

    Energy Technology Data Exchange (ETDEWEB)

    Mohamed, F. [School of Physics, University of Exeter, Exeter EX44QL (United Kingdom)], E-mail: f.mohamed@ex.ac.uk; Bradley, D.A. [Department of Physics, University of Surrey, Guildford GU72XH (United Kingdom); Winlove, C.P. [School of Physics, University of Exeter, Exeter EX44QL (United Kingdom)

    2007-09-21

    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, {alpha}=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, {alpha}=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, {alpha}=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{sup -1} and dose of 75 Gy), again suggesting depolymerization.

  8. Extracellular matrix as a driver for lung regeneration.

    Science.gov (United States)

    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.

  9. C/EBP β Mediates Endoplasmic Reticulum Stress Regulated Inflammatory Response and Extracellular Matrix Degradation in LPS-Stimulated Human Periodontal Ligament Cells

    Directory of Open Access Journals (Sweden)

    Yudi Bai

    2016-03-01

    Full Text Available Periodontitis is an oral inflammatory disease that not only affects the integrity of local tooth-supporting tissues but also impacts systemic health. A compositional shift in oral microbiota has been considered as the main cause of periodontitis; however, the potential mechanism has not been fully defined. Herein, we investigated the role of CCAAT/enhancer-binding protein β (C/EBP β, a member of the C/EBP family of transcription factors, in human periodontal ligament cells (hPDLCs exposed to Porphyromonas gingivalis (P. gingivalis lipopolysaccharide (LPS. RT-PCR and Western blotting analysis showed that the expression of C/EBP β was significantly increased in hPDLCs stimulated with LPS stimuli. Overexpression of C/EBP β by the recombinant adenoviral vector pAd/C/EBP β markedly increased the expression of the pro-inflammatory cytokines IL-6 and IL-8, and matrix metalloproteinases (MMP-8 and -9 in hPDLCs in response to LPS. Furthermore, the activation of endoplasmic reticulum (ER stress was confirmed in LPS-stimulated hPDLCs by measuring the expression of the ER stress marker molecules protein kinase-like ER kinase (PERK, eIF2α, GRP78/Bip, and C/EBP homologous protein (CHOP. The ER stress inhibitor salubrinal repressed, but inducer tunicamycin enhanced, the production of IL-6, IL-8, MMP-8, and MMP-9 in hPDLCs. Additionally, ER stress inducer tunicamycin significantly increased the expression level of C/EBP β in hPDLCs. Blocking of C/EBP β by siRNA resulted in a significant decrease in the secretion of IL-6 and IL-8 and expression of MMP-8 and MMP-9 induced by tunicamycin treatment in hPDLCs. Taken together, ER stress appears to play a regulatory role in the inflammatory response and extracellular matrix (ECM degradation in hPDLCs in response to LPS stimuli by activating C/EBP β expression. This enhances our understanding of human periodontitis pathology.

  10. C/EBP β Mediates Endoplasmic Reticulum Stress Regulated Inflammatory Response and Extracellular Matrix Degradation in LPS-Stimulated Human Periodontal Ligament Cells.

    Science.gov (United States)

    Bai, Yudi; Wei, Yi; Wu, Lian; Wei, Jianhua; Wang, Xiaojing; Bai, Yuxiang

    2016-03-22

    Periodontitis is an oral inflammatory disease that not only affects the integrity of local tooth-supporting tissues but also impacts systemic health. A compositional shift in oral microbiota has been considered as the main cause of periodontitis; however, the potential mechanism has not been fully defined. Herein, we investigated the role of CCAAT/enhancer-binding protein β (C/EBP β), a member of the C/EBP family of transcription factors, in human periodontal ligament cells (hPDLCs) exposed to Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). RT-PCR and Western blotting analysis showed that the expression of C/EBP β was significantly increased in hPDLCs stimulated with LPS stimuli. Overexpression of C/EBP β by the recombinant adenoviral vector pAd/C/EBP β markedly increased the expression of the pro-inflammatory cytokines IL-6 and IL-8, and matrix metalloproteinases (MMP)-8 and -9 in hPDLCs in response to LPS. Furthermore, the activation of endoplasmic reticulum (ER) stress was confirmed in LPS-stimulated hPDLCs by measuring the expression of the ER stress marker molecules protein kinase-like ER kinase (PERK), eIF2α, GRP78/Bip, and C/EBP homologous protein (CHOP). The ER stress inhibitor salubrinal repressed, but inducer tunicamycin enhanced, the production of IL-6, IL-8, MMP-8, and MMP-9 in hPDLCs. Additionally, ER stress inducer tunicamycin significantly increased the expression level of C/EBP β in hPDLCs. Blocking of C/EBP β by siRNA resulted in a significant decrease in the secretion of IL-6 and IL-8 and expression of MMP-8 and MMP-9 induced by tunicamycin treatment in hPDLCs. Taken together, ER stress appears to play a regulatory role in the inflammatory response and extracellular matrix (ECM) degradation in hPDLCs in response to LPS stimuli by activating C/EBP β expression. This enhances our understanding of human periodontitis pathology.

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

    OpenAIRE

    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.

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

    Science.gov (United States)

    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. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. The gene expression of human endothelial cells is modulated by subendothelial extracellular matrix proteins: short-term response to laminar shear stress.

    Science.gov (United States)

    Chlupac, Jaroslav; Filova, Elena; Havlikova, Jana; Matejka, Roman; Riedel, Tomas; Houska, Milan; Brynda, Eduard; Pamula, Elzbieta; Rémy, Murielle; Bareille, Reine; Fernandez, Philippe; Daculsi, Richard; Bourget, Chantal; Bacakova, Lucie; Bordenave, Laurence

    2014-08-01

    Vascular surgery for atherosclerosis is confronted by the lack of a suitable bypass material. Tissue engineering strives to produce bio-artificial conduits to provide resistance to thrombosis. The objectives of our study were to culture endothelial cells (EC) on composite assemblies of extracellular matrix proteins, and to evaluate the cellular phenotype under flow. Cell-adhesive assemblies were fabricated on glass slides as combinations of collagen (Co), laminin (LM), and fibronectin (FN), resulting in three samples: Co, Co/LM, and Co/FN. Surface topography, roughness, and wettability were determined. Human saphenous vein EC were harvested from cardiac patients, cultured on the assemblies and submitted to laminar shear stress (SS) of 12 dyn/cm(2) for 40, 80, and 120 min. Cell retention was assessed and qRT-PCR of adhesion genes (VE-cadherin, vinculin, KDR, CD-31 or PECAM-1, β1-integrins) and metabolic genes (t-PA, NF-κB, eNOS and MMP-1) was performed. Quantitative immunofluorescence of VE cadherin, vinculin, KDR, and vonWillebrand factor was performed after 2 and 6 h of flow. Static samples were excluded from shearing. The cells reached confluence with similar growth curves. The cells on Co/LM and Co/FN were resistant to flow up to 120 min but minor desquamation occurred on Co corresponding with temporary downregulation of VE cadherin and vinculin-mRNA and decreased fluorescence of vinculin. The cells seeded on Co/LM initially more upregulated vinculin-mRNA and also the inflammatory factor NF-κB, and the cells plated on Co/FN changed the expression profile minimally in comparison with the static control. Fluorescence of VE cadherin and vonWillebrand factor was enhanced on Co/FN. The cells cultured on Co/LM and Co/FN increased the vinculin fluorescence and expressed more VE cadherin and KDR-mRNA than the cells on Co. The cells plated on Co/FN upregulated the mRNA of VE cadherin, CD-31, and MMP 1 to a greater extent than the cells on Co/LM and they

  14. From mechanotransduction to extracellular matrix gene expression in fibroblasts.

    Science.gov (United States)

    Chiquet, Matthias; Gelman, Laurent; Lutz, Roman; Maier, Silke

    2009-05-01

    Tissue mechanics provide an important context for tissue growth, maintenance and function. On the level of organs, external mechanical forces largely influence the control of tissue homeostasis by endo- and paracrine factors. On the cellular level, it is well known that most normal cell types depend on physical interactions with their extracellular matrix in order to respond efficiently to growth factors. Fibroblasts and other adherent cells sense changes in physical parameters in their extracellular matrix environment, transduce mechanical into chemical information, and integrate these signals with growth factor derived stimuli to achieve specific changes in gene expression. For connective tissue cells, production of the extracellular matrix is a prominent response to changes in mechanical load. We will review the evidence that integrin-containing cell-matrix adhesion contacts are essential for force transmission from the extracellular matrix to the cytoskeleton, and describe novel experiments indicating that mechanotransduction in fibroblasts depends on focal adhesion adaptor proteins that might function as molecular springs. We will stress the importance of the contractile actin cytoskeleton in balancing external with internal forces, and describe new results linking force-controlled actin dynamics directly to the expression of specific genes, among them the extracellular matrix protein tenascin-C. As assembly lines for diverse signaling pathways, matrix adhesion contacts are now recognized as the major sites of crosstalk between mechanical and chemical stimuli, with important consequences for cell growth and differentiation.

  15. A titanium surface with nano-ordered spikes and pores enhances human dermal fibroblastic extracellular matrix production and integration of collagen fibers.

    Science.gov (United States)

    Yamada, Masahiro; Kato, Eiji; Yamamoto, Akiko; Sakurai, Kaoru

    2016-02-02

    The acquisition of substantial dermal sealing determines the prognosis of percutaneous titanium-based medical devices or prostheses. A nano-topographic titanium surface with ordered nano-spikes and pores has been shown to induce periodontal-like connective tissue attachment and activate gingival fibroblastic functions. This in vitro study aimed to determine whether an alkali-heat (AH) treatment-created nano-topographic titanium surface could enhance human dermal fibroblastic functions and binding strength to the deposited collagen on the titanium surface. The surface topographies of commercially pure titanium machined discs exposed to two different AH treatments were evaluated. Human dermal fibroblastic cultures grown on the discs were evaluated in terms of cellular morphology, proliferation, extracellular matrix (ECM) and proinflammatory cytokine synthesis, and physicochemical binding strength of surface-deposited collagen. An isotropically-patterned, shaggy nano-topography with a sponge-like inner network and numerous well-organized, anisotropically-patterned fine nano-spikes and pores were observed on each nano-topographic surface type via scanning electron microscopy. In contrast to the typical spindle-shaped cells on the machined surfaces, the isotropically- and anisotropically-patterned nano-topographic titanium surfaces had small circular/angular cells containing contractile ring-like structures and elongated, multi-shaped cells with a developed cytoskeletal network and multiple filopodia and lamellipodia, respectively. These nano-topographic surfaces enhanced dermal-related ECM synthesis at both the protein and gene levels, without proinflammatory cytokine synthesis or reduced proliferative activity. Deposited collagen fibers were included in these surfaces and sufficiently bound to the nano-topographies to resist the physical, enzymatic and chemical detachment treatments, in contrast to machined surfaces. Well-organized, isotropically

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

    Directory of Open Access Journals (Sweden)

    Hass Jamie L

    2012-01-01

    Full Text Available Abstract 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

  17. Control of extracellular matrix assembly by syndecan-2 proteoglycan

    DEFF Research Database (Denmark)

    Klass, C M; Couchman, J R; Woods, A

    2000-01-01

    Extracellular matrix (ECM) deposition and organization is maintained by transmembrane signaling and integrins play major roles. We now show that a second transmembrane component, syndecan-2 heparan sulfate proteoglycan, is pivotal in matrix assembly. Chinese Hamster Ovary (CHO) cells were stably...

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

    Energy Technology Data Exchange (ETDEWEB)

    An, Steven S., E-mail: san@jhsph.edu [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Kim, Jina [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Ahn, Kwangmi [Division of Biostatistics, Penn State College of Medicine, Hershey, PA 17033 (United States); Trepat, Xavier [CIBER, Enfermedades Respiratorias, 07110 Bunyola (Spain); Drake, Kenneth J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Fredberg, Jeffrey J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Biswal, Shyam [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205 (United States)

    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.

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

    Science.gov (United States)

    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

    2010-01-01

    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. PMID:19327344

  20. Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer

    OpenAIRE

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

    2009-01-01

    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 E...

  1. Vitamin A Deficiency and Alterations in the Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Teresa Barber

    2014-11-01

    Full Text Available 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.

  2. Syndecans as receptors and organizers of the extracellular matrix

    DEFF Research Database (Denmark)

    Xian, Xiaojie; Gopal, Sandeep; Couchman, John

    2009-01-01

    and signalling molecules, such as protein kinases. Some aspects of syndecan signalling are understood but much remains to be learned. The functions of syndecans in regulating cell adhesion and extracellular matrix assembly are described here. Evidence from null mice suggests that syndecans have roles......, the collagens and glycoproteins of the extracellular matrix are prominent. Frequently, they do so in conjunction with other receptors, most notably the integrins. For this reason, they are often referred to as "co-receptors". However, just as with integrins, syndecans can interact with actin-associated proteins...

  3. The PI3K/Akt Signaling Pathway Mediates the High Glucose-Induced Expression of Extracellular Matrix Molecules in Human Retinal Pigment Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Dong Qin

    2015-01-01

    Full Text Available Prolonged hyperglycemia is an important risk factor of the pathogenesis of diabetic retinopathy (DR. Extracellular matrix molecules, such as fibronectin, collagen IV, and laminin, are associated with fibrotic membranes. In this study, we investigated the expression of fibronectin, collagen IV, and laminin in RPE cells under high glucose conditions. Furthermore, we also detected the phosphorylation of protein kinase B (Akt under high glucose conditions in RPE cells. Our results showed that high glucose upregulated fibronectin, collagen IV, and laminin expression, and activated Akt in RPE cells. We also found that pretreatment with LY294002 (an inhibitor of phosphatidylinositol 3-kinase abolished high glucose-induced expression of fibronectin, collagen IV, and laminin in RPE cells. Thus, high glucose induced the expression of fibronectin, collagen IV, and laminin through PI3K/Akt signaling pathway in RPE cells, and the PI3K/Akt signaling pathway may contribute to the formation of fibrotic membrane during the development of DR.

  4. Reconstruction of rabbit urethra using urethral extracellular matrix

    Institute of Scientific and Technical Information of China (English)

    杨嗣星; 姚颐; 胡云飞; 宋超; 王玲珑; 金化民

    2004-01-01

    Background Urethral reconstruction for both congenital and acquired etiologies remains a challenge for most urologic surgeons. Tissue engineering has been proposed as a strategy for urethral reconstruction. The purpose of This study was to determine whether a naturally derived extracellular matrix substitute developed for urethral reconstruction would be suitable for urethral repair in an animal model.Methods A urethral segmental defect was created in 20 male rabbits. The urethral extracellular matrix, obtained and processed from rabbit urethral tissue, was trimmed and transplanted to repair the urethral defect. Then, the regenerated segment was studied histologically by haematoxylin-eosin staining and Van Gieson staining at 10 days, 3 weeks, 6 weeks, and 24 weeks postoperation. Retrograde urethrography was used to evaluate the function of the regenerated urethras of 4 rabbits 10 and 24 weeks after the operation. The urodynamics of 4 rabbits from the experimental group and control group Ⅰ were assessed and compared. In addition, 4 experimental group rabbits were examined by a urethroscope 24 weeks after the operation.Results At 10 days after operation, epithelial cells had migrated from each side, and small vessels were observed in the extracellular matrix. The matrix and adjacent areas of the host tissue were infiltrated with inflammatory cells. The epithelium covered the extracellular matrix fully at 3 weeks postoperation. Well-formed smooth-muscle cells were first confirmed after 6 weeks, at which point the inflammatory cells had disappeared. At 24 weeks postoperation, the regenerated tissue was equivalent to the normal urethra. Urethrography and urodynamic evaluations showed that there was no difference between normal tissue and regenerated tissue.Conclusions Urethral extracellular matrix appears to be a useful material for urethral repair in rabbits. The matrix can be processed easily and has good characteristics for tissue handling and urethral function.

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

    Science.gov (United States)

    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.

  6. Extracellular Matrix Assembly in Diatoms (Bacillariophyceae)1

    Science.gov (United States)

    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

  7. Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms.

    Science.gov (United States)

    Klein, Marlise I; Hwang, Geelsu; Santos, Paulo H S; Campanella, Osvaldo H; Koo, Hyun

    2015-01-01

    Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases.

  8. Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms

    Directory of Open Access Journals (Sweden)

    Marlise eKlein

    2015-02-01

    Full Text Available Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS, eDNA and lipoteichoic acid (LTA. EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases.

  9. A Look inside the Listeria monocytogenes Biofilms Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Angelo Colagiorgi

    2016-07-01

    Full Text Available 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.

  10. Organ-specific chemotactic factors present in lung extracellular matrix.

    Science.gov (United States)

    Cerra, R F; Nathanson, S D

    1989-05-01

    The preferential colonization of a distant organ by a circulating tumor cell (organ specific metastasis) may be regulated by chemotactic factors present within the extracellular matrix of the host organ. Organ-specific extracellular matrix was prepared from murine kidney and lung by high salt extraction and DNAase/RNAase digestion. A soluble protein fraction (S2) from each of the matricies was obtained by 4 M guanidine extraction and was tested for organ-specific chemotactic activity in a modified Boyden chamber. The lung colonizing B16-F10 and B16-BL6 tumor cell lines demonstrated organ-specific motility only toward the lung extract. The low metastasizing B16 parental line and liver colonizing B16-L4b line showed no preference for either lung or kidney. The lung activity resolves into five fractions by gel filtration chromatography, with the highest activity eluting at Mr approximately 71,000. Chemotactic factors present in lung extracellular matrix may regulate the preferential colonization of an organ by stimulating the migration of tumor cells in a specific manner. These factors may be released during the degradation of the extracellular matrix.

  11. Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

    NARCIS (Netherlands)

    Blaauboer, M.E.; Boeijen, F.R.; Emson, C.L.; Turner, S.M.; Zandieh-Doulabi, B.; Hanemaaijer, R.; Smit, T.H.; Stoop, R.; Everts, V.

    2014-01-01

    Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the

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

    Science.gov (United States)

    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. Airway smooth muscle - Its relationship to the extracellular matrix

    NARCIS (Netherlands)

    Black, Judith L.; Burgess, Janette K.; Johnson, Peter R.A.

    2003-01-01

    The airway smooth muscle cell has a variety of properties, which confer on it the ability to participate actively in the inflammatory process and the remodeling events, which accompany severe, persistent asthma. Among these properties is its relationship to the extracellular matrix (ECM) with which

  14. Basic Components of Connective Tissues and Extracellular Matrix

    DEFF Research Database (Denmark)

    Halper, Jaroslava; Kjær, Michael

    2014-01-01

    of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its...... network. Laminins contribute to the structure of the extracellular matrix (ECM) and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin...... and TGFβ activity was observed in fibrotic skin disorders such as keloids and scleroderma. Cartilage oligomeric matrix protein (COMP) or thrombospondin-5 is primarily present in the cartilage. High levels of COMP are present in fibrotic scars and systemic sclerosis of the skin, and in tendon, especially...

  15. Extracellular matrix in canine mammary tumors with special focus on versican, a versatile extracellular proteoglycan

    NARCIS (Netherlands)

    Erdélyi, Ildikó

    2006-01-01

    The extracellular matrix (ECM) research has become fundamental to understand cancer. This thesis focuses on the exploration of ECM composition and organization in canine mammary tumors, with a special interest in the large chondroitin-sulfate proteoglycan (PG), versican. Chapter 1 gives an overvie

  16. Extracellular matrix components direct porcine muscle stem cell behavior

    Energy Technology Data Exchange (ETDEWEB)

    Wilschut, Karlijn J. [Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Haagsman, Henk P. [Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht (Netherlands); Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl [Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht (Netherlands)

    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. OASIS/CREB3L1 is induced by endoplasmic reticulum stress in human glioma cell lines and contributes to the unfolded protein response, extracellular matrix production and cell migration.

    Directory of Open Access Journals (Sweden)

    Ravi N Vellanki

    Full Text Available OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines (U373, A172 and U87 and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells.

  18. Modification of the secretion pattern of proteases, inflammatory mediators, and extracellular matrix proteins by human aortic valve is key in severe aortic stenosis.

    Science.gov (United States)

    Alvarez-Llamas, Gloria; Martín-Rojas, Tatiana; de la Cuesta, Fernando; Calvo, Enrique; Gil-Dones, Felix; Dardé, Veronica M; Lopez-Almodovar, Luis F; Padial, Luis R; Lopez, Juan-Antonio; Vivanco, Fernando; Barderas, Maria G

    2013-09-01

    One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation. Degenerative aortic stenosis is the most common worldwide cause of valve replacement. Using a proteomic analysis of the secretome from aortic stenosis valves we could identify candidate markers related to this pathology, which may facilitate early diagnosis and treatment. For this purpose, we have designed a method to validate the origin of secreted proteins, demonstrating their synthesis and release by the tissue and ruling out blood origin. The nLC-MS/MS analysis showed the labeling of 61 proteins, 82% of which incorporated the label in only one group. Western blot and selective reaction monitoring differential analysis, revealed a notable role of the extracellular matrix. Variation in particular proteins such as PEDF, cystatin and clusterin emphasizes the link between aortic stenosis and atherosclerosis. In particular, certain proteins variation in secretome levels correlates well, not only with label incorporation trend (only labeled in aortic stenosis group) but, more importantly, with alterations found in plasma from an independent cohort of samples, pointing to specific candidate markers to follow up in diagnosis, prognosis, and therapeutic intervention.

  19. Matricryptins and matrikines: biologically active fragments of the extracellular matrix.

    Science.gov (United States)

    Ricard-Blum, Sylvie; Salza, Romain

    2014-07-01

    Numerous extracellular proteins and glycosaminoglycans (GAGs) undergo limited enzymatic cleavage resulting in the release of fragments exerting biological activities, which are usually different from those of the full-length molecules. In this review, we define matrikines and matricryptins, which are bioactive fragments released from the extracellular matrix proteins, proteoglycans and GAGs and report their major biological activities. These fragments regulate a number of physiopathological processes including angiogenesis, cancer, fibrosis, inflammation, neurodegenerative diseases and wound healing. The challenges to translate these fragments from molecules biologically active in vitro and in experimental models to potential drugs are discussed in the last part of the review.

  20. IMMUNOHISTOCHEMICAL STUDY OF EXTRACELLULAR-MATRIX IN ACUTE GALACTOSAMINE HEPATITIS IN RATS

    NARCIS (Netherlands)

    JONKER, AM; DIJKHUIS, FWJ; BOES, A; HARDONK, MJ

    1992-01-01

    A single injection of D-galactosamine hydrochloride induces acute self-limiting liver disease in rats that morphologically resembles drug-induced hepatitis in human beings. In this immunohistochemical study we examined the localization and expression of the hepatic extracellular matrix components fi

  1. Lung protection by inhalation of exogenous solubilized extracellular matrix

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  3. Expression of genes encoding extracellular matrix proteins: a macroarray study.

    Science.gov (United States)

    Futyma, Konrad; Miotła, Paweł; Różyńska, Krystyna; Zdunek, Małgorzata; Semczuk, Andrzej; Rechberger, Tomasz; Wojcierowski, Jacek

    2014-12-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  7. Targeting the extracellular matrix to disrupt cancer progression

    OpenAIRE

    Freja Albjerg Venning; Lena eWullkopf; Janine T. Erler

    2015-01-01

    Metastatic complications are responsible for more than 90% of cancer related deaths. The progression from an isolated tumor to disseminated metastatic disease is a multi-step process, with each step involving intricate cross-talk between the cancer cells and their non-cellular surroundings, the extracellular matrix (ECM). Many ECM proteins are significantly de-regulated during the progression of cancer, causing both biochemical and biomechanical changes that together promote the metastatic ca...

  8. Stroma and extracellular matrix proteins in canine tumours

    OpenAIRE

    2004-01-01

    In this thesis, studies on temporal and spatial changes in stromal cells and extracellular matrix (ECM) molecules in canine gastrointestinal (GIT) tumours and canine transmissible venereal (CTVT) tumours are described. The mechanisms involved in the phenotypic transformation of fibroblasts to myofibroblasts, and ECM changes were investigated. We found that the myofibroblast is the most common stromal cell in canine GIT epithelial tumours and most likely originated from pre-existing fibroblast...

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

    Directory of Open Access Journals (Sweden)

    Abigail Hielscher

    Full Text Available 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.

  10. Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture*

    OpenAIRE

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

    2009-01-01

    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...

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

    Science.gov (United States)

    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.

  12. The molecular elasticity of the extracellular matrix protein tenascin

    Science.gov (United States)

    Oberhauser, Andres F.; Marszalek, Piotr E.; Erickson, Harold P.; Fernandez, Julio M.

    1998-05-01

    Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were ~25nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain,. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain,.

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

    Science.gov (United States)

    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

  14. Cellular Traction Stresses Mediate Extracellular Matrix Degradation by Invadopodia

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  16. Biglycan fragmentation in pathologies associated with extracellular matrix remodeling by matrix metalloproteinases

    DEFF Research Database (Denmark)

    Genovese, Federica; Barascuk, Natasha; Larsen, Lise Skakkebæk;

    2013-01-01

    The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragmentation is likely to be associated with collagen turnover during the pathogenesis of diseases which involve dysregulated extracellular matrix remodeling (ECMR), such as rheumatoid arthritis (RA) and liver fibrosis...

  17. Experimental study on urethral reconstruction with extracellular matrix of human umbillical vein%胎儿脐静脉脱细胞基质构建组织工程尿道

    Institute of Scientific and Technical Information of China (English)

    范应中; 王海江; 张谦; 张大; 李泸平; 文建国; 王焱

    2010-01-01

    Objective To assess the feasibility of using extracellular matrix (ECM) of human umbilical veins in reconstruction of urethral defect.Methods Extracellular matrix of human umbilical veins was prepared using hypotonic,hypertonic solutions,detergent and proteinase in a multistep process.Twenty-four male rabbits were divided randomly into three groups,including experimental group A,control group B and C.In group A(n=12),,the 2.0cm urethral defect was repaired with extracellular matrix of human umbilical vein graft.The regeneration of urethra was assessed with histology,urodynamic study and retrograde urethrography post-operatively.Results Morphological analyses showed that the cells were removed effectively from umbilical veins and the elastic and collagen fibers were.Two weeks post-operatively,,epithelial cells migrated from each side into the ECM.Minimal infihration of inflammatory cells was also observed at the host-matrix anastomosis;however,these cells disappeared 8 weeks postoperatively.The epithelium covered the DCM fully 4 weeks postoperatively.At 8th post-operative week,minimal extracellular matrix of Human umbilical veins graft was observed in the host tissue.New smooth-muscle cells were confirmed.Sixteen weeks postopera-tively,the histology of group A is similar to that of the group C,and there was no difference in the number of the urethral smooth muscles between the tWO groups.But the smooth muscles of group A were obviously disorganized comparing with those of group C The urehrography and urodynarnic evalnation revealed that there was no difference between the group A and the group C Conclusions Extracellular matrix of human umbilieal veins is potentially a good candidate material for urethral reconstruction.%目的 探讨人脐静脉细胞外基质的制备及其在尿道重建中的应用.方法 采用酶消化、去污剂和渗透溶液方法制备人脐静脉细胞外基质(extracellular matrix,ECM).24只雄性白兔,随机分为A尿道ECM移植组,

  18. Recent advances in the study of zebrafish extracellular matrix proteins.

    Science.gov (United States)

    Jessen, Jason R

    2015-05-01

    The zebrafish extracellular matrix (ECM) is a dynamic and pleomorphic structure consisting of numerous proteins that together regulate a variety of cellular and morphogenetic events beginning as early as gastrulation. The zebrafish genome encodes a similar complement of ECM proteins as found in other vertebrate organisms including glycoproteins, fibrous proteins, proteoglycans, glycosaminoglycans, and interacting or modifying proteins such as integrins and matrix metalloproteinases. As a genetic model system combined with its amenability to high-resolution microscopic imaging, the zebrafish allows interrogation of ECM protein structure and function in both the embryo and adult. Accumulating data have identified important roles for zebrafish ECM proteins in processes as diverse as cell polarity, migration, tissue mechanics, organ laterality, muscle contraction, and regeneration. In this review, I highlight recently published data on these topics that demonstrate how the ECM proteins fibronectin, laminin, and collagen contribute to zebrafish development and adult homeostasis.

  19. Three-dimensional reconstruction of skeletal muscle extracellular matrix ultrastructure.

    Science.gov (United States)

    Gillies, Allison R; Bushong, Eric A; Deerinck, Thomas J; Ellisman, Mark H; Lieber, Richard L

    2014-12-01

    The skeletal muscle extracellular matrix (ECM) supports muscle's passive mechanical function and provides a unique environment for extracellular tissues such as nerves, blood vessels, and a cadre of mononuclear cells. Within muscle ECM, collagen is thought to be the primary load-bearing protein, yet its structure and organization with respect to muscle fibers, tendon, and mononuclear cells is unknown. Detailed examination of extracellular collagen morphology requires high-resolution electron microscopy performed over relatively long distances because multinucleated muscle cells are very long and extend from several millimeters to several centimeters. Unfortunately, there is no tool currently available for high resolution ECM analysis that extends over such distances relevant to muscle fibers. Serial block face scanning electron microscopy is reported here to examine skeletal muscle ECM ultrastructure over hundreds of microns. Ruthenium red staining was implemented to enhance contrast and utilization of variable pressure imaging reduced electron charging artifacts, allowing continuous imaging over a large ECM volume. This approach revealed previously unappreciated perimysial collagen structures that were reconstructed via both manual and semi-automated segmentation methods. Perimysial collagen structures in the ECM may provide a target for clinical therapies aimed at reducing skeletal muscle fibrosis and stiffness.

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

    Science.gov (United States)

    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. More than 70 papers have been published on extracellular matrix (ECM) hydrogels created from source tissue in almost every organ system. The present manuscript represents a review of ECM hydrogels and attempts to identify structure-function relationships that influence the tissue remodeling outcomes and gaps in the understanding thereof. There is a Phase 1 clinical trial now in progress for an ECM hydrogel. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Veiga S.S.

    2001-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  3. Extracellular matrix of the bovine ovarian membrana granulosa.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  8. Detection of extracellular genomic DNA scaffold in human thrombus

    DEFF Research Database (Denmark)

    Oklu, Rahmi; Albadawi, Hassan; Watkins, Michael T

    2012-01-01

    PURPOSE: Mechanisms underlying transition of a thrombus susceptible to tissue plasminogen activator (TPA) fibrinolysis to one that is resistant is unclear. Demonstration of a new possible thrombus scaffold may open new avenues of research in thrombolysis and may provide mechanistic insight...... thrombi. CONCLUSIONS: Extensive detection of genomic DNA associated with histones in the extracellular matrix of human and mouse thrombi suggest the presence of a new thrombus-associated scaffold....

  9. Accelerated extracellular matrix turnover during exacerbations of COPD

    DEFF Research Database (Denmark)

    Sand, Jannie M B; Knox, Alan J; Lange, Peter

    2015-01-01

    BACKGROUND: Exacerbations of chronic obstructive pulmonary disease (COPD) contribute significantly to disease progression. However, the effect on tissue structure and turnover is not well described. There is an urgent clinical need for biomarkers of disease activity associated with disease...... progression. Extracellular matrix (ECM) turnover reflects activity in tissues and consequently assessment of ECM turnover may serve as biomarkers of disease activity. We hypothesized that the turnover of lung ECM proteins were altered during exacerbations of COPD. METHODS: 69 patients with COPD hospitalised......, respectively), and degradation of elastin (ELM7 and EL-NE) and versican (VCANM). RESULTS: Circulating levels of C3M, C4M, C6M, ELM7, and EL-NE were elevated during an exacerbation of COPD as compared to follow-up (all P

  10. Extracellular matrix adaptation of tendon and skeletal muscle to exercise

    DEFF Research Database (Denmark)

    Kjaer, Michael; Magnusson, Peter; Krogsgaard, Michael

    2006-01-01

    The extracellular matrix (ECM) of connective tissues enables linking to other tissues, and plays a key role in force transmission and tissue structure maintenance in tendons, ligaments, bone and muscle. ECM turnover is influenced by physical activity, and both collagen synthesis and metalloprotease......-beta and IL-6 is enhanced following exercise. For tendons, metabolic activity (e.g. detected by positron emission tomography scanning), circulatory responses (e.g. as measured by near-infrared spectroscopy and dye dilution) and collagen turnover are markedly increased after exercise. Tendon blood flow...... is supported by findings of gender-related differences in the activation of collagen synthesis with exercise. These findings may provide the basis for understanding tissue overloading and injury in both tendons and skeletal muscle....

  11. Targeting the extracellular matrix to disrupt cancer progression

    Directory of Open Access Journals (Sweden)

    Freja Albjerg Venning

    2015-10-01

    Full Text Available Metastatic complications are responsible for more than 90% of cancer related deaths. The progression from an isolated tumor to disseminated metastatic disease is a multi-step process, with each step involving intricate cross-talk between the cancer cells and their non-cellular surroundings, the extracellular matrix (ECM. Many ECM proteins are significantly de-regulated during the progression of cancer, causing both biochemical and biomechanical changes that together promote the metastatic cascade. In this review, the influence of several ECM proteins on these multiple steps of cancer spread is summarized. In addition, we highlight the promising (pre-clinical data showing benefits of targeting these ECM macromolecules to prevent cancer progression.

  12. Characterization of canine platelet adhesion to extracellular matrix proteins.

    Science.gov (United States)

    Pelagalli, Alessandra; Pero, Maria Elena; Mastellone, Vincenzo; Cestaro, Anna; Signoriello, Simona; Lombardi, Pietro; Avallone, Luigi

    2011-07-01

    Canine platelets have been extensively studied but little is known about specific aspects such as adhesion. Platelet adhesion is a critical step during haemostasis and thrombosis as well as during inflammatory and immunopathogenic responses. The aim of this study was to evaluate the adhesive properties of canine platelets using fibrinogen and collagen as substrates immobilized on plates. Adhesion was monitored for 120 min and the effect of adenosine 5'-diphosphate (ADP) was assayed. The results showed that canine platelets displayed good adhesion activity that was significantly time-dependent. Moreover, ADP was able to enhance platelet adhesion in a dose-dependent manner. The findings aid knowledge of the adhesion process and suggest a specific role of surface platelet receptors in mediating the interaction with extracellular matrix proteins.

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

    Directory of Open Access Journals (Sweden)

    Jessica L Eisenberg

    2011-01-01

    Full Text Available Jessica L Eisenberg1,2, Asmahan Safi3, Xiaoding Wei3, Horacio D Espinosa3, GR Scott Budinger2, Desire Takawira1, Susan B Hopkinson1, Jonathan CR Jones1,21Department of Cell and Molecular Biology, 2Division of Pulmonary Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA; 3Department of Mechanical Engineering, Northwestern University, Evanston, IL, USAAim: 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.Keywords: alveolar epithelial cells, fibrosis, extracellular matrix, substrate stiffness

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

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, Larissa Ferraz [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Mataveli, Fábio D’Aguiar [Universidade Federal de São Paulo, São Paulo, SP (Brazil); Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva [Universidade Federal de São Paulo, São Paulo, SP (Brazil)

    2015-07-01

    Evaluate the effects of VEGF{sub 165} gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. 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 VEGF{sub 165} 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. 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 VEGF{sub 165}. 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 VEGF{sub 165}, suggesting greater tissue differentiation. The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF{sub 165} seems to provide a protective effect in the treatment of acute myocardial infarct.

  15. Extracellular matrix assembly and organization during zebrafish gastrulation.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  17. Effect of human extracellular matrix protein 1 on proliferation of MCF-7 and human umbilical vein endothelial cells%细胞外基质蛋白1对MCF-7和HUVEC增殖影响的研究

    Institute of Scientific and Technical Information of China (English)

    侯彦强; 蔡丽君; 范列英; 罗鹏; 何玮; 娄加陶; 周演武; 倪健; 孔宪涛; 仲人前

    2004-01-01

    目的:探讨细胞外基质蛋白1(extracellular matrix protein 1,ECM1)对乳腺癌细胞株MCF-7和人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)增殖的影响.方法:构建ECM1-pEGFP-N2真核表达载体,采用PCR方法,扩增出ECM1基因,用BglⅡ和Kpn Ⅰ双酶切ECM1基因和pEGFP-N2载体,连接酶切目的片段,转化大肠杆菌DH5α,筛选阳性克隆酶切、测序鉴定;利用脂质体介导的转染技术转染MCF-7细胞,药物G418筛选稳定转染细胞株,荧光显微镜检测报告基因表达产物EGFP,免疫组化检测ECM1蛋白表达.用MTT比色法分析ECM1对MCF-7和HUVEC增殖的影响.结果:成功构建了ECM1-pEGFP-N2真核表达载体,并在MCF-7中稳定表达;MTT比色法检测MCF-7增殖结果显示未转染组、空载体转染组和ECM1转染组的D570值分别为0.95±0.07、0.97±0.09和1.03±0.12,三者无明显差异;MTT比色法检测HUVEC增殖结果示培养液组、空载体转染上清组、ECM1转染上清组HUVEC D570值分别为0.89±0.06、0.92±0.09和1.39±0.10,差异具有显著性意义(P<0.01).结论:ECM1对乳腺癌细胞株MCF-7的增殖无影响,但能显著促进血管内皮细胞体外增殖.

  18. Tumor necrosis factor-α and receptor activator of nuclear factor-κB ligand augment human macrophage foam-cell destruction of extracellular matrix through protease-mediated processes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Larsen, Lise;

    2012-01-01

    component of extracellular matrix (ECM) in plaques, and to establish whether the pro-inflammatory molecules, tumor necrosis factor (TNF)-alpha, and receptor activator of nuclear factor-κB ligand (RANK-L) increase this degradation. CD14+ monocytes isolated from peripheral blood were differentiated......% and 72%, respectively. This is, to our knowledge, the first data describing a simple in vitro system in which macrophage foam cells degradation of matrix proteins can be monitored. This degradation can be enhanced by cytokines since TNF-alpha and RANK-L significantly increased the matrix degradation...

  19. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Directory of Open Access Journals (Sweden)

    Jun-Ha Hwang

    Full Text Available Mesenchymal stem cell (MSC differentiation is regulated by the extracellular matrix (ECM through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  20. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Science.gov (United States)

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  3. A novel assay for extracellular matrix remodeling associated with liver fibrosis

    DEFF Research Database (Denmark)

    Barascuk, N; Veidal, S S; Larsen, L

    2010-01-01

    Accumulation of extracellular matrix (ECM) components and increased matrix-metalloprotease (MMPs) activity are hallmarks of fibrosis. We developed an ELISA for quantification of MMP-9 derived collagen type III (CO3) degradation....

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Karen Stanic

    2016-09-01

    Full Text Available Extracellular matrix (ECM molecules are pivotal for central nervous system development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axonal 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 central nervous system 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 towards 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.

  6. DMPD: Fragments of extracellular matrix as mediators of inflammation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18243041 Fragments of extracellular matrix as mediators of inflammation. Adair-Kirk...l) Show Fragments of extracellular matrix as mediators of inflammation. PubmedID 18243041 Title Fragments of extrace... TL, Senior RM. Int J Biochem Cell Biol. 2008;40(6-7):1101-10. Epub 2007 Dec 24. (.png) (.svg) (.html) (.csm

  7. Association between pro-inflammatory cytokine expression, angiogenesis, extracellular matrix remodeling, and prognosis in cervical cancer

    NARCIS (Netherlands)

    Zijlmans, Henry Johanna Maria Antonius Adrianus

    2014-01-01

    Growth and progression of cervical carcinoma is dependent on a complex interaction between cervical carcinoma cells and composition of the extracellular matrix. For local progression as well as metastasizing, the extracellular matrix needs to be rearranged creating space for tumor cells to expand an

  8. Astrocytes as a source for Extracellular matrix molecules and cytokines

    Directory of Open Access Journals (Sweden)

    Stefan eWiese

    2012-06-01

    Full Text Available 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. Deeper knowledge of the differentiation processes during development of the central nervous system (CNS might help explaining and even help treating neurological diseases like Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS and psychiatric disorders in which astrocytes have been shown to play a role. Astrocytes and oligodendrocytes develop from a multipotent stem cell that prior to this has produced primarily neuronal precursor cells. This switch towards the more astroglial differentiation is regulated by a change in receptor composition on the cell surface and responsiveness of the respective trophic factors Fibroblast growth factor (FGF and Epidermal growth factor (EGF. The glial precursor cell is driven into the astroglial direction by signaling molecules like Ciliary neurotrophic factor (CNTF, Bone Morphogenetic Proteins (BMPs, 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. On the other hand, ECM molecules expressed by reactive astrocytes are also known to act mostly in an inhibitory fashion under pathophysiological conditions. In this regard, we further summarize recent data concerning the role of chondroitin sulfate proteoglycans and Tnc under pathological

  9. C/EBP β Mediates Endoplasmic Reticulum Stress Regulated Inflammatory Response and Extracellular Matrix Degradation in LPS-Stimulated Human Periodontal Ligament Cells

    OpenAIRE

    Yudi Bai; Yi Wei; Lian Wu; Jianhua Wei; Xiaojing Wang; Yuxiang Bai

    2016-01-01

    Periodontitis is an oral inflammatory disease that not only affects the integrity of local tooth-supporting tissues but also impacts systemic health. A compositional shift in oral microbiota has been considered as the main cause of periodontitis; however, the potential mechanism has not been fully defined. Herein, we investigated the role of CCAAT/enhancer-binding protein β (C/EBP β), a member of the C/EBP family of transcription factors, in human periodontal ligament cells (hPDLCs) exposed t...

  10. Tissue-Specific Effects of Esophageal Extracellular Matrix.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  13. Extracellular matrix stiffness dictates Wnt expression through integrin pathway.

    Science.gov (United States)

    Du, Jing; Zu, Yan; Li, Jing; Du, Shuyuan; Xu, Yipu; Zhang, Lang; Jiang, Li; Wang, Zhao; Chien, Shu; Yang, Chun

    2016-02-08

    It is well established that extracellular matrix (ECM) stiffness plays a significant role in regulating the phenotypes and behaviors of many cell types. However, the mechanism underlying the sensing of mechanical cues and subsequent elasticity-triggered pathways remains largely unknown. We observed that stiff ECM significantly enhanced the expression level of several members of the Wnt/β-catenin pathway in both bone marrow mesenchymal stem cells and primary chondrocytes. The activation of β-catenin by stiff ECM is not dependent on Wnt signals but is elevated by the activation of integrin/ focal adhesion kinase (FAK) pathway. The accumulated β-catenin then bound to the wnt1 promoter region to up-regulate the gene transcription, thus constituting a positive feedback of the Wnt/β-catenin pathway. With the amplifying effect of positive feedback, this integrin-activated β-catenin/Wnt pathway plays significant roles in mediating the enhancement of Wnt signal on stiff ECM and contributes to the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance. The present integrin-regulated Wnt1 expression and signaling contributes to the understanding of the molecular mechanisms underlying the regulation of cell behaviors by ECM elasticity.

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

    Science.gov (United States)

    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.

  15. [Glycation of extracellular matrix proteins and its role in atherosclerosis].

    Science.gov (United States)

    Kuzan, Aleksandra; Chwiłkowska, Agnieszka; Kobielarz, Magdalena; Pezowicz, Celina; Gamian, Andrzej

    2012-10-29

    Glycation consists in formation of advanced glycation end-products (AGE) during non-enzymatic reaction between reducing sugars and proteins, lipids or nucleic acids. This review is focused mainly on glycation of collagen and its role in acceleration of vascular disease. Collagen is an extracellular matrix protein characterized by unique structure forming fibrils with great anti-tensile and anti-breaking strength. The protein builds the connective tissue and is responsible for biomechanical properties of blood vessels. It is reported that higher content of glycated collagen correlates with lower elasticity and greater toughness of the vessel walls and, as a consequence, a faster rate of atherosclerosis development. Numerous mechanisms connected with AGE formation are involved in atherogenesis, among others: receptor-mediated production of free radicals, triggering an inflammatory process, activation of leukocytes and thrombocytes, facilitation of LDL binding, change in level of growth factors, adhesion molecules, MMP and some other proteins' expression. The coverages allow the development of therapeutic strategies to prevent or slow down the pathological processes connected with glycation of collagen and other proteins in the artery wall. The main strategies are based on limitation of exogenous AGE, consumption of products which contain rutin, treatment with drugs which inhibit AGE formation, such as pyridoxamine, and chemicals which are able to cleave already formed AGE protein-protein crosslinks, such as ALT-711.

  16. Chemical Decellularization Methods and Its Effects on Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Amir Hossein Akbari Zahmati

    2017-08-01

    Full Text Available Background:  Extracellular matrix (ECM produced by tissue decellularization processes as a biological scaffold due to its unique properties compared to other scaffolds for migration and implantation of stem cells have been used successfully in the field of tissue engineering and regenerative medicine in the last years. The objective of this manuscript was to provide an overview of the chemical decellularization methods, evaluation of decellularized ECM and the potential effect of the chemical decellularization agents on the biochemical composition. Methods: We searched in Google Scholar, PubMed, Scopus, and Science Direct. The literature search was done by using the following keywords: “ECM, biologic scaffold, decellularization, chemical methods, tissue engineering.” We selected articles have been published from 2000 to 2016, and 15 full texts and 97 abstracts were reviewed. Results:Employing an optimization method to minimize damage to the ECM ultrastructure as for a result of the lack of reduction in mechanical properties and also the preservation of essential proteins such as laminin, fibronectin, Glycosaminoglycans (GAGs, growth factor is required. Various methods include chemical, physical and enzymatic technics were studied. However, on each of these methods can have undesirable effects on ECM. Conclusion: It is suggested that instead of the Sodium dodecyl sulfate (SDS which have high strength degradation, we can use zwitterionic separately or in combination with SDS. Tributyl phosphate (TBP due to its unique properties can be used in decellularization process.

  17. Inflammatory Alterations of the Extracellular Matrix in the Tumor Microenvironment

    Energy Technology Data Exchange (ETDEWEB)

    Iijima, Junko [Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-Ku, Kyoto 603-8555 (Japan); Konno, Kenjiro [Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-Ku, Kyoto 603-8555 (Japan); Itano, Naoki, E-mail: itanon@cc.kyoto-su.ac.jp [Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-Ku, Kyoto 603-8555 (Japan)

    2011-08-09

    Complex interactions between cancer cells and host stromal cells result in the formation of the “tumor microenvironment”, where inflammatory alterations involve the infiltration of tumor-associated fibroblasts and inflammatory leukocytes that contribute to the acquisition of malignant characteristics, such as increased cancer cell proliferation, invasiveness, metastasis, angiogenesis, and avoidance of adaptive immunity. The microenvironment of a solid tumor is comprised not only of cellular compartments, but also of bioactive substances, including cytokines, growth factors, and extracellular matrix (ECM). ECM can act as a scaffold for cell migration, a reservoir for cytokines and growth factors, and a signal through receptor binding. During inflammation, ECM components and their degraded fragments act directly and indirectly as inflammatory stimuli in certain cases and regulate the functions of inflammatory and immune cells. One such ECM component, hyaluronan, has recently been implicated to modulate innate immune cell function through pattern recognition toll-like receptors and accelerate the recruitment and activation of tumor-associated macrophages in inflamed cancers. Here, we will summarize the molecular mechanism linking inflammation with ECM remodeling in the tumor microenvironment, with a particular emphasis on the role of hyaluronan in controlling the inflammatory response.

  18. Collagens and proteoglycans of the corneal extracellular matrix

    Directory of Open Access Journals (Sweden)

    Y.M. Michelacci

    2003-08-01

    Full Text Available The cornea is a curved and transparent structure that provides the initial focusing of a light image into the eye. It consists of a central stroma that constitutes 90% of the corneal depth, covered anteriorly with epithelium and posteriorly with endothelium. Its transparency is the result of the regular spacing of collagen fibers with remarkably uniform diameter and interfibrillar space. Corneal collagen is composed of heterotypic fibrils consisting of type I and type V collagen molecules. The cornea also contains unusually high amounts of type VI collagen, which form microfibrillar structures, FACIT collagens (XII and XIV, and other nonfibrillar collagens (XIII and XVIII. FACIT collagens and other molecules, such as leucine-rich repeat proteoglycans, play important roles in modifying the structure and function of collagen fibrils.Proteoglycans are macromolecules composed of a protein core with covalently linked glycosaminoglycan side chains. Four leucine-rich repeat proteoglycans are present in the extracellular matrix of corneal stroma: decorin, lumican, mimecan and keratocan. The first is a dermatan sulfate proteoglycan, and the other three are keratan sulfate proteoglycans. Experimental evidence indicates that the keratan sulfate proteoglycans are involved in the regulation of collagen fibril diameter, and dermatan sulfate proteoglycan participates in the control of interfibrillar spacing and in the lamellar adhesion properties of corneal collagens. Heparan sulfate proteoglycans are minor components of the cornea, and are synthesized mainly by epithelial cells. The effect of injuries on proteoglycan synthesis is discussed.

  19. Brain extracellular matrix retains connectivity in neuronal networks.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Adaptive evolution of the matrix extracellular phosphoglycoprotein in mammals.

    Science.gov (United States)

    Machado, João Paulo; Johnson, Warren E; O'Brien, Stephen J; Vasconcelos, Vítor; Antunes, Agostinho

    2011-11-21

    Matrix extracellular phosphoglycoprotein (MEPE) belongs to a family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs) that play a key role in skeleton development, particularly in mineralization, phosphate regulation and osteogenesis. MEPE associated disorders cause various physiological effects, such as loss of bone mass, tumors and disruption of renal function (hypophosphatemia). The study of this developmental gene from an evolutionary perspective could provide valuable insights on the adaptive diversification of morphological phenotypes in vertebrates. Here we studied the adaptive evolution of the MEPE gene in 26 Eutherian mammals and three birds. The comparative genomic analyses revealed a high degree of evolutionary conservation of some coding and non-coding regions of the MEPE gene across mammals indicating a possible regulatory or functional role likely related with mineralization and/or phosphate regulation. However, the majority of the coding region had a fast evolutionary rate, particularly within the largest exon (1467 bp). Rodentia and Scandentia had distinct substitution rates with an increased accumulation of both synonymous and non-synonymous mutations compared with other mammalian lineages. Characteristics of the gene (e.g. biochemical, evolutionary rate, and intronic conservation) differed greatly among lineages of the eight mammalian orders. We identified 20 sites with significant positive selection signatures (codon and protein level) outside the main regulatory motifs (dentonin and ASARM) suggestive of an adaptive role. Conversely, we find three sites under selection in the signal peptide and one in the ASARM motif that were supported by at least one selection model. The MEPE protein tends to accumulate amino acids promoting disorder and potential phosphorylation targets. MEPE shows a high number of selection signatures, revealing the crucial role of positive selection in the evolution of this SIBLING member. The selection

  3. Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

    LENUS (Irish Health Repository)

    Davis, Niall F

    2011-04-01

    To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

  4. Glycation of extracellular matrix proteins impairs migration of immune cells.

    Science.gov (United States)

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells.

  5. Microtubules regulate GEF-H1 in response to extracellular matrix stiffness

    Science.gov (United States)

    Heck, Jessica N.; Ponik, Suzanne M.; Garcia-Mendoza, Maria G.; Pehlke, Carolyn A.; Inman, David R.; Eliceiri, Kevin W.; Keely, Patricia J.

    2012-01-01

    Breast epithelial cells sense the stiffness of the extracellular matrix through Rho-mediated contractility. In turn, matrix stiffness regulates RhoA activity. However, the upstream signaling mechanisms are poorly defined. Here we demonstrate that the Rho exchange factor GEF-H1 mediates RhoA activation in response to extracellular matrix stiffness. We demonstrate the novel finding that microtubule stability is diminished by a stiff three-dimensional (3D) extracellular matrix, which leads to the activation of GEF-H1. Surprisingly, activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway did not contribute to stiffness-induced GEF-H1 activation. Loss of GEF-H1 decreases cell contraction of and invasion through 3D matrices. These data support a model in which matrix stiffness regulates RhoA through microtubule destabilization and the subsequent release and activation of GEF-H1. PMID:22593214

  6. The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

    Science.gov (United States)

    Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

    2012-07-01

    Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells.

  7. Extracellular matrix structure and nano-mechanics determine megakaryocyte function.

    Science.gov (United States)

    Malara, Alessandro; Gruppi, Cristian; Pallotta, Isabella; Spedden, Elise; Tenni, Ruggero; Raspanti, Mario; Kaplan, David; Tira, Maria Enrica; Staii, Cristian; Balduini, Alessandra

    2011-10-20

    Cell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-α2β1-dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis.

  8. Extracellular matrix of dental pulp stem cells: Applications in pulp tissue engineering using somatic MSCs

    Directory of Open Access Journals (Sweden)

    Sriram eRavindran

    2014-01-01

    Full Text Available Dental Caries affects approximately 90% of the world’s population. At present, the clinical treatment for dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality. Tissue engineering can potentially solve this problem by enabling regeneration of a functional pulp tissue. Dental pulp stem cells (DPSCs have been shown to be an excellent source for pulp regeneration. However, limited availability of these cells hinders its potential for clinical translation. We have investigated the possibility of using somatic mesenchymal stem cells from other sources for dental pulp tissue regeneration using a biomimetic dental pulp extracellular matrix (ECM incorporated scaffold. Human periodontal ligament stem cells (PDLSCs and human bone marrow stromal cells (HMSCs were investigated for their ability to differentiate towards an odontogenic lineage. In vitro real-time PCR results coupled with histological and immunohistochemical examination of the explanted tissues confirmed the ability of PDLSCs and HMSCs to form a vascularized pulp-like tissue. These findings indicate that the dental pulp stem derived ECM scaffold stimulated odontogenic differentiation of PDLSCs and HMSCs without the need for exogenous addition of growth and differentiation factors. This study represents a translational perspective toward possible therapeutic application of using a combination of somatic stem cells and extracellular matrix for pulp regeneration.

  9. Adaptive evolution of the matrix extracellular phosphoglycoprotein in mammals

    Directory of Open Access Journals (Sweden)

    Machado João

    2011-11-01

    Full Text Available Abstract Background Matrix extracellular phosphoglycoprotein (MEPE belongs to a family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs that play a key role in skeleton development, particularly in mineralization, phosphate regulation and osteogenesis. MEPE associated disorders cause various physiological effects, such as loss of bone mass, tumors and disruption of renal function (hypophosphatemia. The study of this developmental gene from an evolutionary perspective could provide valuable insights on the adaptive diversification of morphological phenotypes in vertebrates. Results Here we studied the adaptive evolution of the MEPE gene in 26 Eutherian mammals and three birds. The comparative genomic analyses revealed a high degree of evolutionary conservation of some coding and non-coding regions of the MEPE gene across mammals indicating a possible regulatory or functional role likely related with mineralization and/or phosphate regulation. However, the majority of the coding region had a fast evolutionary rate, particularly within the largest exon (1467 bp. Rodentia and Scandentia had distinct substitution rates with an increased accumulation of both synonymous and non-synonymous mutations compared with other mammalian lineages. Characteristics of the gene (e.g. biochemical, evolutionary rate, and intronic conservation differed greatly among lineages of the eight mammalian orders. We identified 20 sites with significant positive selection signatures (codon and protein level outside the main regulatory motifs (dentonin and ASARM suggestive of an adaptive role. Conversely, we find three sites under selection in the signal peptide and one in the ASARM motif that were supported by at least one selection model. The MEPE protein tends to accumulate amino acids promoting disorder and potential phosphorylation targets. Conclusion MEPE shows a high number of selection signatures, revealing the crucial role of positive selection in the

  10. Data from acellular human heart matrix

    Directory of Open Access Journals (Sweden)

    Pedro L Sánchez

    2016-09-01

    Full Text Available Perfusion decellularization of cadaveric hearts removes cells and generates a cell-free extracellular matrix scaffold containing acellular vascular conduits, which are theoretically sufficient to perfuse and support tissue-engineered heart constructs. This article contains additional data of our experience decellularizing and testing structural integrity and composition of a large series of human hearts, “Acellular human heart matrix: a critical step toward whole heat grafts” (Sanchez et al., 2015 [1]. Here we provide the information about the heart decellularization technique, the valve competence evaluation of the decellularized scaffolds, the integrity evaluation of epicardial and myocardial coronary circulation, the pressure volume measurements, the primers used to assess cardiac muscle gene expression and, the characteristics of donors, donor hearts, scaffolds and perfusion decellularization process.

  11. Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia.

    Science.gov (United States)

    McKee, Marc D; Hoac, Betty; Addison, William N; Barros, Nilana M T; Millán, José L; Chaussain, Catherine

    2013-10-01

    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 for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) 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 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 and phosphate-regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X-linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X-linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating small integrin-binding ligand N-linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine- and aspartate-rich-motif peptide, may accumulate locally to impair mineralization in this disease.

  12. Neutrophils contribute to fracture healing by synthesizing fibronectin+ extracellular matrix rapidly after injury

    NARCIS (Netherlands)

    Bastian, Okan W.; Koenderman, Leo; Alblas, Jacqueline; Leenen, Luke P H; Blokhuis, Taco J.

    2016-01-01

    The role of inflammatory cells in bone regeneration remains unclear. We hypothesize that leukocytes contribute to fracture healing by rapidly synthesizing an "emergency extracellular matrix (ECM)" before stromal cells infiltrate the fracture hematoma (FH) and synthesize the eventual collagenous bone

  13. Extracellular matrix proteins modulate asthmatic airway smooth muscle cell proliferation via an autocrine mechanism

    NARCIS (Netherlands)

    Johnson, Peter R A; Burgess, Janette K; Underwood, P Anne; Au, Wendy; Poniris, Maree H; Tamm, Michael; Ge, Qi; Roth, Michael; Black, Judith L

    2004-01-01

    BACKGROUND: Airway remodeling is a key feature of persistent asthma and includes alterations in the extracellular matrix protein profile around the airway smooth muscle (ASM) and hyperplasia of the ASM. We have previously shown that nonasthmatic ASM cells in culture produce a range of extracellular

  14. First pregnancies, live birth, and in vitro fertilization outcomes after transplantation of frozen-banked ovarian tissue with a human extracellular matrix scaffold using robot-assisted minimally invasive surgery.

    Science.gov (United States)

    Oktay, Kutluk; Bedoschi, Giuliano; Pacheco, Fernanda; Turan, Volkan; Emirdar, Volkan

    2016-01-01

    Ovarian tissue cryopreservation is an experimental fertility preservation method and the transplantation techniques are still evolving. We attempted to improve the technique with the utility of a human decellularized extracellular tissue matrix (ECTM) scaffold, robot-assisted minimally invasive surgery, and perioperative pharmacological support. We prospectively studied 2 subjects with hemophagocytic lymphohistiocytosis (patient A) and non-Hodgkin lymphoma (patient B) who underwent ovarian tissue cryopreservation at the age of 23 years, before receiving preconditioning chemotherapy for hematopoietic stem cell transplantation. Both experienced ovarian failure postchemotherapy and we transplanted ovarian cortical tissues to the contralateral menopausal ovary 7 and 12 years later, using a human ECTM scaffold and robotic assistance. The ECTM scaffold tissue compatibility was shown in preclinical studies. Patients also received estrogen supplementation and baby aspirin preoperatively to aid in the revascularization process. Ovarian follicle development was observed approximately 10 (patient A) and 8 (patient B) weeks after ovarian tissue transplantation. Following 8 and 7 cycles of in vitro fertilization, 9 and 10 day-3 embryos were cryopreserved (patients A and B, respectively). While the baseline follicle-stimulating hormone (range 3.6-15.4 mIU/mL) levels near normalized by 7 months and remained steady postovarian transplantation in patient A, patient B showed improved but elevated follicle-stimulating hormone levels throughout (range 21-31 mIU/mL). Highest follicle yield was achieved 14 (8 follicles; patient A) and 11 (6 follicles; patient B) months postintervention. Patient A experienced a chemical pregnancy after the third frozen embryo transfer attempt. She then conceived following her first fresh in vitro fertilization embryo transfer and the pregnancy is currently ongoing. Patient B conceived after the first frozen embryo transfer attempt and delivered a

  15. Fibulin-1 is a marker for arterial extracellular matrix alterations in type 2 diabetes

    DEFF Research Database (Denmark)

    Cangemi, Claudia; Skov, Vibe; Poulsen, Michael Kjaer

    2011-01-01

    Extracellular matrix alterations are important elements in the arterial changes seen in diabetes, being associated with increased vascular stiffness and the development of cardiovascular diseases. However, no biomarkers for diabetes-related arterial changes have been defined.......Extracellular matrix alterations are important elements in the arterial changes seen in diabetes, being associated with increased vascular stiffness and the development of cardiovascular diseases. However, no biomarkers for diabetes-related arterial changes have been defined....

  16. The Effects of Extracellular Matrix on Tissue Engineering Construction of Cartilage in Vitro

    Institute of Scientific and Technical Information of China (English)

    YU Li; LI Fa-tao; TANG Ming-qiao; YAN Wei-qun

    2006-01-01

    The effects of various cartilage extracellular matrix on the construction of rabbit growth plate cartilage tissue in vitro were studied. The results show that collagen, proteoglycan and hyaluronic acid can promote the growth of cultured chondrocytes but the effects of various cartilage extracellular matrix(ECM)on chondrocyte differentiation are different. Collagen can promote the hypertrophy of chondrocytes while proteoglycan and hyaluronic acid inhibit the transition of mature chondrocytes into hypertrophied chondrocytes.

  17. The extracellular matrix of the lung and its role in edema formation

    OpenAIRE

    Paolo Pelosi; Rocco, Patricia R. M.; Daniela Negrini; Alberto Passi

    2007-01-01

    The extracellular matrix is composed of a three-dimensional fiber mesh filled with different macromolecules such as: collagen (mainly type I and III), elastin, glycosaminoglycans, and proteoglycans. In the lung, the extracellular matrix has several functions which provide: 1) mechanical tensile and compressive strength and elasticity, 2) low mechanical tissue compliance contributing to the maintenance of normal interstitial fluid dynamics, 3) low resistive pathway for an effective gas exchang...

  18. Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

    OpenAIRE

    Hermann, M.; Jaconi, M E; Dahlgren, C; Waldvogel, F A; Stendahl, O; Lew, D P

    1990-01-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (m...

  19. Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells.

    Science.gov (United States)

    Haage, Amanda; Schneider, Ian C

    2014-08-01

    The pathogenesis of cancer is often driven by local invasion and metastasis. Recently, mechanical properties of the tumor microenvironment have been identified as potent regulators of invasion and metastasis, while matrix metalloproteinases (MMPs) are classically known as significant enhancers of cancer cell migration and invasion. Here we have been able to sensitively measure MMP activity changes in response to specific extracellular matrix (ECM) environments and cell contractility states. Cells of a pancreatic cancer cell line, Panc-1, up-regulate MMP activities between 3- and 10-fold with increased cell contractility. Conversely, they down-regulate MMP activities when contractility is blocked to levels seen with pan-MMP activity inhibitors. Similar, albeit attenuated, responses are seen in other pancreatic cancer cell lines, BxPC-3 and AsPC-1. In addition, MMP activity was modulated by substrate stiffness, collagen gel concentration, and the degree of collagen cross-linking, when cells were plated on collagen gels ranging from 0.5 to 5 mg/ml that span the physiological range of substrate stiffness (50-2000 Pa). Panc-1 cells showed enhanced MMP activity on stiffer substrates, whereas BxPC-3 and AsPC-1 cells showed diminished MMP activity. In addition, eliminating heparan sulfate proteoglycans using heparinase completely abrogated the mechanical induction of MMP activity. These results demonstrate the first functional link between MMP activity, contractility, and ECM stiffness and provide an explanation as to why stiffer environments result in enhanced cell migration and invasion.

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

    Science.gov (United States)

    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.

  1. Extracellular matrix remodelling in myocardial hypertrophy and failure : focus on osteopontin.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  3. Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix.

    Science.gov (United States)

    Vincourt, Jean-Baptiste; Etienne, Stéphanie; Grossin, Laurent; Cottet, Justine; Bantsimba-Malanda, Claudie; Netter, Patrick; Mainard, Didier; Libante, Virginie; Gillet, Pierre; Magdalou, Jacques

    2012-06-01

    The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.

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

    Science.gov (United States)

    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.

  5. The regulatory roles of small leucine-rich proteoglycans in extracellular matrix assembly.

    Science.gov (United States)

    Chen, Shoujun; Birk, David E

    2013-05-01

    Small leucine-rich proteoglycans (SLRPs) are involved in a variety of biological and pathological processes. This review focuses on their regulatory roles in matrix assembly. SLRPs have protein cores and hypervariable glycosylation with multivalent binding abilities. During development, differential interactions of SLRPs with other molecules result in tissue-specific spatial and temporal distributions. The changing expression patterns play a critical role in the regulation of tissue-specific matrix assembly and therefore tissue function. SLRPs play significant structural roles within extracellular matrices. In addition, they play regulatory roles in collagen fibril growth, fibril organization and extracellular matrix assembly. Moreover, they are involved in mediating cell-matrix interactions. Abnormal SLRP expression and/or structures result in dysfunctional extracellular matrices and pathophysiology. Altered expression of SLRPs has been found in many disease models, and structural deficiency also causes altered matrix assembly. SLRPs regulate assembly of the extracellular matrix, which defines the microenvironment, modulating both the extracellular matrix and cellular functions, with an impact on tissue function.

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

    Science.gov (United States)

    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.

  7. Extracellular Matrix components regulate cellular polarity and tissue structure in the developing and mature Retina

    Directory of Open Access Journals (Sweden)

    Shweta Varshney

    2015-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  10. Cell-extracellular matrix and cell-cell adhesion are linked by syndecan-4

    DEFF Research Database (Denmark)

    Pakideeri Karat, Sandeep Gopal; Multhaupt, Hinke A B; Pocock, Roger

    2017-01-01

    Cell-extracellular matrix (ECM) and cell-cell junctions that employ microfilaments are sites of tension. They are important for tissue repair, morphogenetic movements and can be emblematic of matrix contraction in fibrotic disease and the stroma of solid tumors. One cell surface receptor, syndeca...

  11. Regulation and use of the extracellular matrix by Trypanosoma cruzi during early infection

    Directory of Open Access Journals (Sweden)

    Pius N. Nde

    2012-11-01

    Full Text Available Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global becoming a new worldwide challenge. For more than a century since its discovery, it has remained neglected with no effective drugs or vaccines. The mechanisms by which Trypanosoma cruzi regulates and uses the extracellular matrix to invade cells and cause disease are just beginning to be understood. Here we critically review and discuss the regulation of the extracellular matrix (ECM interactome by T. cruzi, the use of the ECM by T. cruzi and analyze the molecular ECM/T. cruzi interphase during the early process of infection. It has been shown that invasive trypomastigote forms of T. cruzi use and modulate components of the ECM during the initial process of infection. Infective trypomastigotes up-regulate the expression of laminin γ-1 (LAMC1 and thrombospondin (THBS1 to facilitate the recruitment of trypomastigotes to enhance cellular infection. Silencing the expression of LAMC1 and THBS1 by stable RNAi dramatically reduces trypanosome infection. T. cruzi gp83, a ligand that mediates the attachment of trypanosomes to cells to initiate infection, up-regulates LAMC1 expression to enhance cellular infection. Infective trypomastigotes use Tc85 to interact with laminin, p45 mucin to interact with LAMC1 through galectin-3 (LGALS3, a human lectin, and calreticulin (TcCRT to interact with TSB1 to enhance cellular infection. Silencing the expression of LGALS3 also reduces cellular infection. Despite the role of the ECM in T. cruzi infection, almost nothing is known about the ECM interactome networks operating in the process of T. cruzi infection and its ligands. Here, we present the first elucidation of the human ECM interactome network regulated by T. cruzi and its gp83 ligand that facilitates cellular infection. The elucidation of the human ECM interactome regulated by T. cruzi and the dissection of the molecular ECM/T. cruzi interphase using

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

    Science.gov (United States)

    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.

  13. Force-induced unfolding of fibronectin in the extracellular matrix of living cells.

    Directory of Open Access Journals (Sweden)

    Michael L Smith

    2007-10-01

    Full Text Available Whether mechanically unfolded fibronectin (Fn is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other, or from the force-induced unfolding of type III modules (unfolding model. Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes.

  14. Preoperative radiotherapy and extracellular matrix remodeling in rectal mucosa and tumour matrix metalloproteinases and plasminogen components

    Energy Technology Data Exchange (ETDEWEB)

    Angenete, Eva; Oeresland, Tom; Falk, Peter; Breimer, Michael; Ivarsson, Marie-Louise (Dept. of Surgery, Inst. of Clinical Sciences, Sahlgrenska Academy at Univ. of Goeteborg, Goeteborg (Sweden)); Hultborn, Ragnar (Dept. of Oncology, Institute of Clinical Sciences, Sahlgrenska Univ. Hospital/Sahlgrenska, Goeteborg (Sweden))

    2009-11-15

    Background. Preoperative radiotherapy reduces recurrence but increases postoperative morbidity. The aim of this study was to explore the effect of radiotherapy in rectal mucosa and rectal tumour extracellular matrix (ECM) by studying enzymes and growth factors involved in ECM remodeling. Materials and methods. Twenty patients with short-term preoperative radiotherapy and 12 control patients without radiotherapy were studied. Biopsies from rectal mucosa and tumour were collected prior to radiotherapy and at surgery. Tissue MMP-1, -2, -9, TIMP-1, uPA, PAI-1, TGF-beta1 and calprotectin were determined by ELISA. Biopsies from irradiated and non-irradiated peritoneal areas were also analysed. Results. Radiotherapy increased the tissue levels of MMP-2 and PAI-1 in both the rectal mucosa and tumours while calprotectin and uPA showed an increase only in the mucosa after irradiation. The increase of calprotectin was due to an influx of inflammatory cells as revealed by immunohistochemistry. Prior to irradiation, the tumour tissues had increased levels of MMP-1, -2, -9, total TGF-beta1, uPA, PAI-1 and calprotectin compared to mucosa, while TIMP-1 and the active TGF-beta1 fraction showed no statistical difference. Conclusions. This study indicates a radiation-induced effect on selected ECM remodeling proteases. This reaction may be responsible for early and late morbidity. Interference of this response might reduce these consequences.

  15. In silico analysis suggests interaction between Ebola virus and the extracellular matrix

    Directory of Open Access Journals (Sweden)

    Veljko eVeljkovic

    2015-02-01

    Full Text Available The worst Ebola virus (EV outbreak in history has hit Liberia, Sierra Leone and Guinea hardest and the trendlines in this crisis are grave, and now represents global public health threat concern. Limited therapeutic and/or prophylactic options which are available for humans suffering from Ebola virus disease (EVD further complicate situation. Previous studies suggested that the EV glycoprotein (GP is the main determinant causing structural damage of endothelial cells that triggers the hemorrhagic diathesis, but molecular mechanisms underlying this phenomenon remains elusive. Using the informational spectrum method (ISM, a virtual spectroscopy method for analysis of the protein-protein interactions, the interaction of GP with endothelial extracellular matrix (ECM was investigated. Presented results of this in silico study suggest that Elastin Microfibril Interface Located Proteins (EMILINs are involved in interaction between GP and ECM. This finding could contribute to better understanding of EV/endothelium interaction and its role in pathogenesis, prevention and therapy of EVD.

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

    Science.gov (United States)

    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.

  17. Update of human and mouse matrix metalloproteinase families

    OpenAIRE

    Jackson Brian C; Nebert Daniel W; Vasiliou Vasilis

    2010-01-01

    Abstract Matrix metalloproteinases (MMPs) are a family of zinc proteases that degrade most of the components of the extracellular matrix (ECM). MMPs also have a number of non-traditional roles in processing factors related to cell growth/proliferation, inflammation and more. There are 23 human MMPs and 23 mouse MMPs, most of which share orthology among most vertebrates; other examples have been found in invertebrates and plants. MMPs are named in order of discovery, but also have been grouped...

  18. RAGE-mediated extracellular matrix proteins accumulation exacerbates HySu-induced pulmonary hypertension.

    Science.gov (United States)

    Jia, Daile; He, Yuhu; Zhu, Qian; Liu, Huan; Zuo, Caojian; Chen, Guilin; Yu, Ying; Lu, Ankang

    2017-05-01

    Extracellular matrix (ECM) proteins accumulation contributes to the progression of pulmonary arterial hypertension (PAH), a rare and fatal cardiovascular condition defined by high pulmonary arterial pressure, whether primary, idiopathic, or secondary to other causes. The receptor for advanced glycation end products (RAGE) is constitutively expressed in the lungs and plays an important role in ECM deposition. Nonetheless, the mechanisms by which RAGE mediates ECM deposition/formation in pulmonary arteries and its roles in PAH progression remain unclear. Expression of RAGE and its activating ligands, S100/calgranulins and high mobility group box 1 (HMGB1), were increased in both human and mouse pulmonary arterial smooth muscle cells (PASMCs) under hypoxic conditions and were also strikingly upregulated in pulmonary arteries in hypoxia plus SU5416 (HySu)-induced PAH in mice. RAGE deletion alleviated pulmonary arterial pressure and restrained extracellular matrix accumulation in pulmonary arteries in HySu-induced PAH murine model. Moreover, blocking RAGE activity with a neutralizing antibody in human PASMCs, or RAGE deficiency in mouse PASMCs exposed to hypoxia, suppressed the expression of fibrotic proteins by reducing TGF-β1 expression. RAGE reconstitution in deficient mouse PASMCs restored hypoxia-stimulated TGF-β1 production via ERK1/2 and p38 MAPK pathway activation and subsequently increased ECM protein expression. Interestingly, HMGB1 acting on RAGE, not toll-like receptor 4 (TLR4), induced ECM deposition in PASMCs. Finally, in both idiopathic PAH patients and HySu-induced PAH mice, soluble RAGE (sRAGE) levels in serum were significantly elevated compared to those in controls. Activation of RAGE facilitates the development of hypoxia-induced pulmonary hypertension by increase of ECM deposition in pulmonary arteries. Our results indicate that sRAGE may be a potential biomarker for PAH diagnosis and disease severity, and that RAGE may be a promising target for

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

    Science.gov (United States)

    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.

  20. High levels of the extracellular matrix proteoglycan decorin are associated with inhibition of testicular function

    Science.gov (United States)

    Adam, Marion; Urbanski, Henryk F.; Garyfallou, Vasilios T.; Welsch, Ulrich; Köhn, Frank M.; Schwarzer, J. Ullrich; Strauss, Leena; Poutanen, Matti; Mayerhofer, Artur

    2011-01-01

    Decorin (DCN), a component of the extracellular matrix of the peritubular wall and the interstitial areas of the human testis, can interact with growth factor (GF) signaling, thereby blocking downstream actions of GFs. In the present study the expression and regulation of DCN using both human testes and two experimental animal models, namely the rhesus monkey and mouse, were examined. DCN protein was present in peritubular and interstitial areas of adult human and monkey testes, while it was almost undetectable in adult wild-type mice. Interestingly, the levels and sites of testicular DCN expression in the monkeys were inversely correlated with testicular maturation markers. A strong DCN expression associated with the abundant connective tissue of the interstitial areas in the postnatal through prepubertal phases was observed. In adult and old monkeys the DCN pattern was similar to the one in normal human testes, presenting strong expression at the peritubular region. In the testes of both infertile men and in a mouse model of inflammation associated infertility (aromatase-overexpressing transgenic mice), the fibrotic changes and increased numbers of Tumor necrosis factor (TNF)-α-producing immune cells were shown to be associated with increased production of DCN. Furthermore, studies with human testicular peritubular cells isolated from fibrotic testis indicated that TNF-α significantly increased DCN production. The data, thus, show that an increased DCN level is associated with impaired testicular function, supporting our hypothesis that DCN interferes with paracrine signaling of the testis in health and disease. PMID:22413766

  1. Processing of procollagen III by meprins: new players in extracellular matrix assembly?

    Science.gov (United States)

    Kronenberg, Daniel; Bruns, Bernd C; Moali, Catherine; Vadon-Le Goff, Sandrine; Sterchi, Erwin E; Traupe, Heiko; Böhm, Markus; Hulmes, David J S; Stöcker, Walter; Becker-Pauly, Christoph

    2010-12-01

    Meprins α and β, a subgroup of zinc metalloproteinases belonging to the astacin family, are known to cleave components of the extracellular matrix, either during physiological remodeling or in pathological situations. In this study we present a new role for meprins in matrix assembly, namely the proteolytic processing of procollagens. Both meprins α and β release the N- and C-propeptides from procollagen III, with such processing events being critical steps in collagen fibril formation. In addition, both meprins cleave procollagen III at exactly the same site as the procollagen C-proteinases, including bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family. Indeed, cleavage of procollagen III by meprins is more efficient than by BMP-1. In addition, unlike BMP-1, whose activity is stimulated by procollagen C-proteinase enhancer proteins (PCPEs), the activity of meprins on procollagen III is diminished by PCPE-1. Finally, following our earlier observations of meprin expression by human epidermal keratinocytes, meprin α is also shown to be expressed by human dermal fibroblasts. In the dermis of fibrotic skin (keloids), expression of meprin α increases and meprin β begins to be detected. Our study suggests that meprins could be important players in several remodeling processes involving collagen fiber deposition.

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

    Science.gov (United States)

    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. Cedrol Enhances Extracellular Matrix Production in Dermal Fibroblasts in a MAPK-Dependent Manner

    Science.gov (United States)

    Jin, Mu Hyun; Park, Sun Gyoo; Hwang, Yul-Lye; Lee, Min-Ho; Jeong, Nam-Ji; Roh, Seok-Seon; Lee, Young; Kim, Chang Deok

    2012-01-01

    Background The extracellular matrix (ECM) produced by dermal fibroblasts supports skin structure, and degradation and/or reduced production of ECM are the main causes of wrinkle formation. Objective The aim of this study was to identify the active ingredient that enhances ECM production in dermal fibroblasts. Methods Polarity-based fractionation was used to isolate the active ingredient from natural extracts, and the effects of cedrol (isolated from Pterocarpus indicusirginia) on ECM production in cultured human dermal fibroblasts was investigated by reverse transcription-polymerase chain reaction, enzyme linked immunosorbent assay, and Western blot analysis. Results Cedrol accelerated fibroblast growth in a dose-dependent manner and increased the production of type 1 collagen and elastin. Phosphorylation of p42/44 extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and Akt was markedly increased by cedrol, indicating that enhanced ECM production is linked to activation of intracellular signaling cascades. Conclusion These results indicate that cedrol stimulates ECM production, with possible applications to the maintenance of skin texture. PMID:22363150

  4. Scleraxis is required for cell lineage differentiation and extracellular matrix remodeling during murine heart valve formation in vivo.

    Science.gov (United States)

    Levay, Agata K; Peacock, Jacqueline D; Lu, Yinhui; Koch, Manuel; Hinton, Robert B; Kadler, Karl E; Lincoln, Joy

    2008-10-24

    Heart valve structures, derived from mesenchyme precursor cells, are composed of differentiated cell types and extracellular matrix arranged to facilitate valve function. Scleraxis (scx) is a transcription factor required for tendon cell differentiation and matrix organization. This study identified high levels of scx expression in remodeling heart valve structures at embryonic day 15.5 through postnatal stages using scx-GFP reporter mice and determined the in vivo function using mice null for scx. Scx(-/-) mice display significantly thickened heart valve structures from embryonic day 17.5, and valves from mutant mice show alterations in valve precursor cell differentiation and matrix organization. This is indicated by decreased expression of the tendon-related collagen type XIV, increased expression of cartilage-associated genes including sox9, as well as persistent expression of mesenchyme cell markers including msx1 and snai1. In addition, ultrastructure analysis reveals disarray of extracellular matrix and collagen fiber organization within the valve leaflet. Thickened valve structures and increased expression of matrix remodeling genes characteristic of human heart valve disease are observed in juvenile scx(-/-) mice. In addition, excessive collagen deposition in annular structures within the atrioventricular junction is observed. Collectively, our studies have identified an in vivo requirement for scx during valvulogenesis and demonstrate its role in cell lineage differentiation and matrix distribution in remodeling valve structures.

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

    Science.gov (United States)

    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.

  6. Extracellular matrix remodeling in patients with ischemic chronic heart failure with preserved ejection fraction

    Directory of Open Access Journals (Sweden)

    V. D. Syvolap

    2015-04-01

    Full Text Available Aim. To identify features, relationships between parameters of the extracellular matrix and renal function in 110 patients with ischemic chronic heart failure the activity of collagen metabolism markers (MMP-9, TIMP-1, PICP, cystatin C, structural and functional parameters of the heart were studied using ELISA, echocardiography. Results. It was established that imbalance in the system MMP/TIMP in ischemic heart failure with preserved left ventricular ejection fraction leads to disruption of the extracellular matrix structural functional sufficiency, increases functional failure and is associated with impaired renal function. Conclusion. Correlation analysis showed significant relationships between MMP/TIMP and GFR, cystatin C, indicating that the significant role of extracellular matrix imbalance in the development of renal dysfunction in patients with ischemic chronic heart failure.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Adherence of extracellular matrix components to modified surfaces of titanium alloys

    Science.gov (United States)

    Stelzer, C.; Uhlmann, E.; Meinke, M.; Lademann, J.; Hansen, U.

    2009-04-01

    The adherence of biological materials on metal surfaces is of special importance in biology and medicine. The underlying interactions between surface and biological materials (e.g. extracellular matrix components or cells) are responsible for the application as a medical device. Numerous products are made of pure titanium and titanium alloys. This paper shows the influence of a laser production technology on machined surfaces of TiAl6V4 and the resulting adherence of biological material on the basis of the surface characterisation. In this study, different machined TiAl6V4 surfaces were used for coatings with extracellular matrix components. For this process, different coating with collagen I monomers and a complex mixture of extracellular matrix proteins derived from the dermal-epidermal basement membrane zone were analysed. The efficiency of the coating was analysed by different methods and the results are presented in this paper.

  9. Extracellular Matrix Proteins Mediate HIV-1 gp120 Interactions with α4β7.

    Science.gov (United States)

    Plotnik, David; Guo, Wenjin; Cleveland, Brad; von Haller, Priska; Eng, Jimmy K; Guttman, Miklos; Lee, Kelly K; Arthos, James; Hu, Shiu-Lok

    2017-08-16

    Gut-homing α4β7(high) CD4(+) T lymphocytes have been shown to be preferentially targeted by Human Immunodeficiency Virus-1 (HIV), and are implicated in HIV pathogenesis. Previous studies demonstrated that HIV envelope protein gp120 binds and signals through α4β7, and that this likely contributes to the infection of α4β7(high) T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α4β7 binding. However, lack of α4β7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α4β7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α4β7 interactions. We show that Chinese Hamster Ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that co-purified with gp120. CHO fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to α4β7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to α4β7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion exchange chromatography abrogated V2-independent gp120-α4β7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-α4β7 interactions in a similar manner to CHO fibronectin. These findings provide an explanation for the apparent contradictory observations regarding the gp120-α4β7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology.IMPORTANCE Immune tissues within the gut are severely damaged by HIV, and this plays an important role in the development of AIDS. Integrin α4β7 plays a major role in the trafficking of lymphocytes, including CD4(+) T cells, into gut lymphoid tissues. Previous reports

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Experimental study on adipose tissue engineering with human adipose-derived stem cells and adipose tissue extracellular matrix scaffold%人细胞外基质支架联合脂肪干细胞构建脂肪组织

    Institute of Scientific and Technical Information of China (English)

    察鹏飞; 高建华; 陈阳; 鲁峰

    2012-01-01

    目的 探讨人脂肪组织细胞外基质(ECM)支架联合人脂肪来源干细胞(ADSCs)构建工程化脂肪组织的可行性.方法 以酶消化法从人抽脂术抽吸物脂质部分获取人ADSCs,体外进行多向分化诱导鉴定,并行DiI荧光标记.从抽脂术的脂质部分分离提取人脂肪组织细胞外基质,经过低温冻干、粉碎、灭菌等处理,制备成粉末状,电镜扫描观察表面特征并将其与ADSCs进行黏附实验,探讨其作为支架材料的可行性.收集人ADSCs,以2×109/L的细胞密度与提取的细胞外基质支架复合后移植于裸鼠背部皮下,同鼠对侧背部皮下移植ECM支架和细胞培养液作为对照,每侧移植0.5 ml,共6只实验鼠.8周后取材,称量标本湿重.取出的标本行苏木素-伊红(HE)染色和油红O染色进行定性判断,分析人脂肪组织ECM支架联合人ADSCs构建工程化脂肪组织的能力.结果 从脂肪组织中分离得到人ADSCs和ECM支架.ADSCs在相应的诱导环境下能够分化成为脂肪细胞、骨细胞和软骨细胞.ECM支架电镜扫描和大体观察具有疏松、多孔的结构特征,适合ADSCs的黏附生长.ADSCs与支架相容性良好,黏附率达(89.87±2.59)%,细胞在支架表面可充分伸展生长.体内移植8周后,实验组和对照组都能够形成新生物,湿重比较实验组较对照组重(P<0.05).经HE切片及油红O染色均证实实验组形成成熟的脂肪组织,对照组不能形成脂肪组织.结论 人脂肪组织ECM支架联合人ADSCs在体内能够成功构建成熟的脂肪组织,8周后支架并无明显吸收.%Objective To explore the possibility of building tissue-engineered adipose tissue with human adipose-derived stem cells (ADSCs) and adipose tissue extracellular matrix (ECM) scaffold,and provide experimental basis for clinical application of tissue-engineered adipose tissue for the repair of soft tissue defects.Methods ADSCs were isolated from adipose tissue by liposuction with the

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

    Science.gov (United States)

    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.

  13. Guided extracellular matrix formation from fibroblast cells cultured on bio-inspired configurable multiscale substrata

    Directory of Open Access Journals (Sweden)

    Won-Gyu Bae

    2015-12-01

    Full Text Available Engineering complex extracellular matrix (ECM is an important challenge for cell and tissue engineering applications as well as for understanding fundamental cell biology. We developed the methodology for fabrication of precisely controllable multiscale hierarchical structures using capillary force lithography in combination with original wrinkling technique for the generation of well-defined native ECM-like platforms by culturing fibroblast cells on the multiscale substrata [1]. This paper provides information on detailed characteristics of polyethylene glycol-diacrylate multiscale substrata. In addition, a possible model for guided extracellular matrix formation from fibroblast cells cultured on bio-inspired configurable multiscale substrata is proposed.

  14. Effect of phentolamine on myocardial extracellular matrix of cardiac remodeling in rats

    Institute of Scientific and Technical Information of China (English)

    Yi-Gang Yin; Ru-Zhu Wang; Zhong-Bao Ruan; Li Zhu

    2014-01-01

    Objective:To study the effects of phentolamine on myocardial extracellular matrix of cardiac remodeling induced by norepinephrine in rats.Methods:24SD rats were divided into3 groups randomly: control groups, norepinephrine groups(model groups), norepinephrine+phentolamine groups(treatment groups).Echocardiography was used to detect changes in cardiac structure and function, the level of collagen volume fraction(CVF) and hydroxyproline as well as collagen content were determined in myocardial tissue, matrix metalloproteinases-2 and collagen Ⅰ in myocardial tissue were localized by immunohistochemitry.Results:Compared with control groups, left ventricular hypertrophy in the model group rats, the hydroxyproline content and CVF was significantly higher(P<0.01), and matrix metalloproteinase-2 and collagen Ⅰ protein expression was significantly increased(P<0.01).Phentolamine significantly improved cardiac hypertrophy in treatment group rats, reduced hydroxyproline,CVF, matrix metalloproteinase2 and collagen Ⅰprotein expression(P<0.05).Conclusions:Phentolamine can effectively reduce the incidence of myocardial hypertrophy and myocardial extracellular matrix remodeling inSD rats, and it can ease myocardial extracellular matrix of cardiac remodeling.It may be associated with reduced expression of matrix metalloproteinase2 and collagen Ⅰ in myocardial tissue remodeling.

  15. Extracellular Matrix Ligand and Stiffness Modulate Immature Nucleus Pulposus Cell-Cell Interactions

    Science.gov (United States)

    Gilchrist, Christopher L.; Darling, Eric M.; Chen, Jun; Setton, Lori A.

    2011-01-01

    The nucleus pulposus (NP) of the intervertebral disc functions to provide compressive load support in the spine, and contains cells that play a critical role in the generation and maintenance of this tissue. The NP cell population undergoes significant morphological and phenotypic changes during maturation and aging, transitioning from large, vacuolated immature cells arranged in cell clusters to a sparse population of smaller, isolated chondrocyte-like cells. These morphological and organizational changes appear to correlate with the first signs of degenerative changes within the intervertebral disc. The extracellular matrix of the immature NP is a soft, gelatinous material containing multiple laminin isoforms, features that are unique to the NP relative to other regions of the disc and that change with aging and degeneration. Based on this knowledge, we hypothesized that a soft, laminin-rich extracellular matrix environment would promote NP cell-cell interactions and phenotypes similar to those found in immature NP tissues. NP cells were isolated from porcine intervertebral discs and cultured in matrix environments of varying mechanical stiffness that were functionalized with various matrix ligands; cellular responses to periods of culture were assessed using quantitative measures of cell organization and phenotype. Results show that soft (<720 Pa), laminin-containing extracellular matrix substrates promote NP cell morphologies, cell-cell interactions, and proteoglycan production in vitro, and that this behavior is dependent upon both extracellular matrix ligand and substrate mechanical properties. These findings indicate that NP cell organization and phenotype may be highly sensitive to their surrounding extracellular matrix environment. PMID:22087260

  16. Extracellular matrix ligand and stiffness modulate immature nucleus pulposus cell-cell interactions.

    Directory of Open Access Journals (Sweden)

    Christopher L Gilchrist

    Full Text Available The nucleus pulposus (NP of the intervertebral disc functions to provide compressive load support in the spine, and contains cells that play a critical role in the generation and maintenance of this tissue. The NP cell population undergoes significant morphological and phenotypic changes during maturation and aging, transitioning from large, vacuolated immature cells arranged in cell clusters to a sparse population of smaller, isolated chondrocyte-like cells. These morphological and organizational changes appear to correlate with the first signs of degenerative changes within the intervertebral disc. The extracellular matrix of the immature NP is a soft, gelatinous material containing multiple laminin isoforms, features that are unique to the NP relative to other regions of the disc and that change with aging and degeneration. Based on this knowledge, we hypothesized that a soft, laminin-rich extracellular matrix environment would promote NP cell-cell interactions and phenotypes similar to those found in immature NP tissues. NP cells were isolated from porcine intervertebral discs and cultured in matrix environments of varying mechanical stiffness that were functionalized with various matrix ligands; cellular responses to periods of culture were assessed using quantitative measures of cell organization and phenotype. Results show that soft (<720 Pa, laminin-containing extracellular matrix substrates promote NP cell morphologies, cell-cell interactions, and proteoglycan production in vitro, and that this behavior is dependent upon both extracellular matrix ligand and substrate mechanical properties. These findings indicate that NP cell organization and phenotype may be highly sensitive to their surrounding extracellular matrix environment.

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

    Science.gov (United States)

    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

  18. Extracellular proteolytic enzymes produced by human pathogenic Vibrio species

    Directory of Open Access Journals (Sweden)

    Shin-Ichi eMiyoshi

    2013-11-01

    Full Text Available Bacteria in the genus Vibrio produce extracellular proteolytic enzymes to obtain nutrients via digestion of various protein substrates. However, the enzymes secreted by human pathogenic species have been documented to modulate the bacterial virulence. Several species including Vibrio cholerae and V. vulnificus are known to produce thermolysin-like metalloproteases termed vibriolysin. The vibriolysin from V. vulnificus, a causative agent of serious systemic infection, is a major toxic factor eliciting the secondary skin damage characterized by formation of the hemorrhagic brae. The vibriolysin from intestinal pathogens may play indirect roles in pathogenicity because it can activate protein toxins and hemagglutinin by the limited proteolysis and can affect the bacterial attachment to or detachment from the intestinal surface by degradation of the mucus layer. Two species causing wound infections, V. alginolyticus and V. parahaemolyticus, produce another metalloproteases so-called collagenases. Although the detailed pathological roles have not been studied, the collagenase is potent to accelerate the bacterial dissemination through digestion of the protein components of the extracellular matrix. Some species produce cymotrypsin-like serine proteases, which may also affect the bacterial virulence potential. The intestinal pathogens produce sufficient amounts of the metalloprotease at the small intestinal temperature; however, the metalloprotease production by extra-intestinal pathogens is much higher around the body surface temperature. On the other hand, the serine protease is expressed only in the absence of the metalloprotease.

  19. Extracellular matrix and cell surface as determinants of connective tissue differentiation.

    Science.gov (United States)

    Solursh, M

    1989-09-01

    This paper reviews in vitro studies, largely from the author's laboratory, concerning the conditions that are permissive for the differentiation of limb bud mesenchymal cells into chondrocytes. In high-density cell culture, even in a defined medium, the same normal sequence of events that is found in vivo in developing cartilage is also observed. This system can be used to study heritable disorders in model systems such as in mutant mouse embryos. In addition, single mesenchymal cells can differentiate into hypertrophic chondrocytes in hydrated collagen gel or agarose cultures. A rounded cell shape promotes chondrogenesis, while a flattened cell shape promotes fibroblast differentiation. The actin cytoskeleton is shown to play a central role in regulating connective tissue cell differentiation. By use of such cell culture manipulations, it is now possible to grow large numbers of fibroblastic cells from human biopsy material for storage and to carry out experimental studies after re-expression of chondrogenesis in gel cultures. It is suggested that cytoskeletal-extracellular matrix interactions play a fundamental role in connective tissue differentiation. Matrix receptors might be developmentally regulated and modify epithelial effects on mesenchymal cells. In this way mesenchymal cells differentiate in a highly organized manner in spatial and temporal terms.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2014-06-01

    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.

  2. Serum markers of the extracellular matrix remodeling reflect antifibrotic therapy in bile-duct ligated rats

    OpenAIRE

    Robert eSchierwagen; Sabine eKlein; Diana Julie Leeming; Michaela eGranzow; Mette Juul Nielsen; Tilman eSauerbruch; Aleksander eKrag; Morten A Karlsdal; Jonel eTrebicka

    2013-01-01

    BackgroundProgression of liver fibrosis is characterized by synthesis and degradation of extracellular matrix (ECM). Matrix-metalloproteinases (MMP) cleave collagen fibers at a specific site and thereby generate soluble fragments of ECM (neo-epitopes). The levels of these neo-epitopes might reflect the stage of liver fibrosis and may allow monitoring of anti-fibrotic therapies. Here we analyzed these neo-epitopes as read-out for a liver directed therapy with statins.MethodsBile duct ligation ...

  3. Extracellular RNAs: development as biomarkers of human disease

    Directory of Open Access Journals (Sweden)

    Joseph F. Quinn

    2015-08-01

    Full Text Available Ten ongoing studies designed to test the possibility that extracellular RNAs may serve as biomarkers in human disease are described. These studies, funded by the NIH Common Fund Extracellular RNA Communication Program, examine diverse extracellular body fluids, including plasma, serum, urine and cerebrospinal fluid. The disorders studied include hepatic and gastric cancer, cardiovascular disease, chronic kidney disease, neurodegenerative disease, brain tumours, intracranial haemorrhage, multiple sclerosis and placental disorders. Progress to date and the plans for future studies are outlined.

  4. The effects of extracellular matrix proteins on neutrophil-endothelial interaction--a roadway to multiple therapeutic opportunities.

    Science.gov (United States)

    Padmanabhan, Jagannath; Gonzalez, Anjelica L

    2012-06-01

    Polymorphoneuclear leukocytes or neutrophils, a major component of white blood cells, contribute to the innate immune response in humans. Upon sensing changes in the microenvironment, neutrophils adhere to the vascular wall, migrate through the endothelial cell (EC)-pericyte bilayer, and subsequently through the extracellular matrix to reach the site of inflammation. These cells are capable of destroying microbes, cell debris, and foreign proteins by oxidative and non-oxidative processes. While primarily mediators of tissue homeostasis, there are an increasing number of studies indicating that neutrophil recruitment and transmigration can also lead to host-tissue injury and subsequently inflammation-related diseases. Neutrophil-induced tissue injury is highly regulated by the microenvironment of the infiltrated tissue, which includes cytokines, chemokines, and the provisional extracellular matrix, remodeled through increased vascular permeability and other cellular infiltrates. Thus, investigation of the effects of matrix proteins on neutrophil-EC interaction and neutrophil transmigration may help identify the proteins that induce pro- or anti-inflammatory responses. This area of research presents an opportunity to identify therapeutic targets in inflammation-related diseases. This review will summarize recent literature on the role of neutrophils and the effects of matrix proteins on neutrophil-EC interactions, with focus on three different disease models: 1) atherosclerosis, 2) COPD, and 3) tumor growth and progression. For each disease model, inflammatory molecules released by neutrophils, important regulatory matrix proteins, current anti-inflammatory treatments, and the scope for further research will be summarized.

  5. Role of Substratum Stiffness in Modulating Genes Associated with Extracellular Matrix and Mechanotransducers YAP and TAZ

    Science.gov (United States)

    Raghunathan, Vijay Krishna; Morgan, Joshua T.; Dreier, Britta; Reilly, Christopher M.; Thomasy, Sara M.; Wood, Joshua A.; Ly, Irene; Tuyen, Binh C.; Hughbanks, Marissa; Murphy, Christopher J.; Russell, Paul

    2013-01-01

    Purpose. Primary open-angle glaucoma is characterized by increased resistance to aqueous humor outflow and a stiffer human trabecular meshwork (HTM). Two Yorkie homologues, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif, encoded by WWTR1 (TAZ), are mechanotransducers of the extracellular-microenvironment and coactivators of transcription. Here, we explore how substratum stiffness modulates the YAP/TAZ pathway and extracellular matrix genes in HTM cells and how this may be play a role in the onset and progression of glaucoma. Methods. HTM cells from normal donors were cultured on hydrogels mimicking the stiffness of normal (5 kPa) and glaucomatous (75 kPa) HTM. Changes in expression of YAP/TAZ related genes and steroid responsiveness were determined. Additionally, transglutaminase-2 expression was determined after YAP silencing. Results. YAP and TAZ are both expressed in human trabecular meshwork cells. In vitro, YAP and TAZ were inversely regulated by substratum stiffness. YAP and 14-3-3σ were downregulated to different extents on stiffer substrates; TAZ, tissue transglutaminase (TGM2), and soluble frizzled-related protein-1 (sFRP-1) were significantly upregulated. CTGF expression appeared to be altered differentially by both YAP and TAZ. Myocilin and angiopoietin-like 7 expression in response to dexamethasone was more pronounced on stiffer substrates. We demonstrated a direct effect by YAP on TGM2 when YAP was silenced by small interfering RNA. Conclusions. The expression of YAP/TAZ and ECM-related-genes is impacted on physiologically relevant substrates. YAP was upregulated in cells on softer substrates. Stiffer substrates resulted in upregulation of canonical Wnt modulators, TAZ and sFRP-1, and thus may influence the progression of glaucoma. These results demonstrate the importance of YAP/TAZ in the HTM and suggest their role in glaucoma. PMID:23258147

  6. Structural and contractile proteins. Part E: Extracellular matrix

    Energy Technology Data Exchange (ETDEWEB)

    Cunningham, L.W.

    1987-01-01

    This book contains 20 papers. Some of the titles are: Characterization of Pro-..cap alpha..2(I) Collagen Gene Mutation by Nuclease S/sub 1/ Mapping: Analysis of Cytoplasmic and Nuclear Messenger RNA in Fibroblasts from Patients with Type I Osteogenesis Imperfecta; R-Loop Analysis of Procollagen Messenger RNA from the Assessment of Human Collagen Mutations; and Bone Glycoproteins.

  7. Extracellular DNA as matrix component in microbial biofilms

    DEFF Research Database (Denmark)

    Chiang, Wen-Chi; Tolker-Nielsen, Tim

    2010-01-01

    to various persistent infections in humans and animals, and to a variety of complications in industry, where solid–water interfaces occur. Knowledge about the molecular mechanisms involved in biofilm formation is necessary for creating strategies to control biofilms. Recent studies have shown...

  8. Extracellular matrix proteins and the dynamics of dentin formation.

    Science.gov (United States)

    Butler, William T; Brunn, Jan C; Qin, Chunlin; McKee, Marc D

    2002-01-01

    Dentinogenesis involves controlled reactions that result in conversion of unmineralized predentin to dentin when apatite crystals are formed. This process is dynamic: Maturation events occur within predentin beginning at the proximal layer and progressing to the predentin-dentin (PD) border. One type of controlled reaction is the proteolytic processing of dentin sialophosphoprotein (DSPP) to dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), by cleavage of at least three highly conserved peptide bonds. We postulate that this processing event represents an activation step, resulting in release of DPP, which is active in its effects on formation and growth of apatite crystals. Dentin matrix protein 1 (DPM1), present as a processed fragment (57-kD protein) in bone, is seen in dentin on sodium dodecyl sulfate polyacrylamide gel electrophoresis as one intact protein of 150-200 kD. Anti-57-kD antibodies elicit immunoreactivity in bone, dentin, and cellular cementum. In bone, the reactivity is associated with osteocytes and their cell processes. Similarly, dentin shows reactivity in odontoblasts, predentin, and the odontoblast processes. In summary, the processing of large sialic acid-rich proteins into smaller fragments may be an important part of the controlled conversion of predentin to dentin and osteoid to bone.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  11. Inhaled steroids modulate extracellular matrix composition in bronchial biopsies of COPD patients : a randomized, controlled trial

    NARCIS (Netherlands)

    Kunz, Lisette I Z; Strebus, Jolanda; Budulac, Simona E; Lapperre, Therese S; Sterk, Peter J; Postma, Dirkje S; Mauad, Thais; Timens, Wim; Hiemstra, Pieter S

    2013-01-01

    Rationale: Smoking and inflammation contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD), which involves changes in extracellular matrix. This is thought to contribute to airway remodeling and airflow obstruction. We have previously observed that long-term treatment with

  12. Extracellular matrix regulates enhanced eotaxin expression in asthmatic airway smooth muscle cells

    NARCIS (Netherlands)

    Chan, Vivien; Burgess, Janette K; Ratoff, Jonathan C; O'connor, Brian J; Greenough, Anne; Lee, Tak H; Hirst, Stuart J

    2006-01-01

    RATIONALE: Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with fibronectin and collagen are key features of asthma. Previously, we have reported these ECM proteins enhance ASM synthetic function. OBJECTIVE: We compared ASM cultured from endobronchial bio

  13. Substrates with patterned extracellular matrix and subcellular stiffness gradients reveal local biomechanical responses.

    Science.gov (United States)

    Tseng, Peter; Di Carlo, Dino

    2014-02-26

    A substrate fabrication process is developed to pattern both the extracellular matrix (ECM) and rigidity at sub-cellular spatial resolution. When growing cells on these substrates, it is found that cells respond locally in their cytoskeleton assembly. The presented method allows unique insight into the biological interpretation of mechanical signals, whereas photolithography-based fabrication is amenable to integration with complex microfabricated substructures.

  14. Proteoglycan changes in the extracellular matrix of lung tissue from patients with pulmonary emphysema

    NARCIS (Netherlands)

    van Straaten, JFM; Coers, W; Noordhoek, JA; Flipsen, JTM; Kauffman, HF; Timens, W; Postma, DS

    1999-01-01

    To characterize the changes in the extracellular matrix in smoking-related pulmonary emphysema, we undertook immunohistochemical studies in lung tissues from controls (n = 7), from patients with mild (n = 11) and severe (n = 8) emphysema, and from patients with lung fibrosis (n = 6). We studied coll

  15. The extracellular matrix - the under-recognized element in lung disease?

    NARCIS (Netherlands)

    Burgess, Janette K.; Mauad, Thais; Tjin, Gavin; Karlsson, Jenny C.; Westergren-Thorsson, Gunilla

    2016-01-01

    The lung is composed of airways and lung parenchyma, and the extracellular matrix (ECM) contains the main building blocks of both components. The ECM provides physical support and stability to the lung, and as such it has in the past been regarded as an inert structure. More recent research has

  16. Structural, biochemical, cellular, and functional changes in skeletal muscle extracellular matrix with aging

    DEFF Research Database (Denmark)

    Kragstrup, Tue Wenzel; Kjaer, M; Mackey, A L

    2011-01-01

    The extracellular matrix (ECM) of skeletal muscle is critical for force transmission and for the passive elastic response of skeletal muscle. Structural, biochemical, cellular, and functional changes in skeletal muscle ECM contribute to the deterioration in muscle mechanical properties with aging...

  17. Separation Anxiety: Detachment from the Extracellular Matrix Induces Metabolic Changes that Can Stimulate Tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Magdalena A. Cichon; Derek C. Radisky

    2010-01-01

    @@ One of the earliest stages of tumor progression involves the ability of cells to survive and proliferate when not attached to the extracellular matrix (ECM). New research using a physiologically relevant breast cancer model reveals how separation from the ECM stimulates metabolic changes characteristic of developing tumors.

  18. Changes of β3 Integrins and Extracellular Matrix Proteins in the Endometrium of Unexplained Infertility

    Institute of Scientific and Technical Information of China (English)

    王化丽; 曲陆荣; 何丽霞; 张颐

    1999-01-01

    The purpose of this study was to investigate changes of β3 integrins and extracellular matrix proteins including fibronectin (FN) , laminin (LN) and collagen type Ⅳ (CL type Ⅳ) on the endometrium of secretory phase from 31 fertile women (fertility group)and 34 women with unexplained infertility (infertility group) by a histochemical method. The results were as follows : In glandular epithelium, β3 integrin appeared in the mid secretory phase and continued to late secretory phase in the fertility group, but was not expressed during the secretory phase in the infertility group.Extracellular matrix proteins from the fertility group were expressed more strongly in mid secretory phase than that in the early secretory phase, and were weakest in the late secretory phase. Compared with the fertility group, the levels of extracellular matrix proteins in the infertility group were elevated in the secretory phase. In conclusion: our current study demonstrate that fie integrin and extracellular matrix proteins are expressed at different levels in the endometrium during the menstrual cycle. They are involved in endometrial changes during the menstrual cycle and during the implantation of the blastocyst. Their unusual expression result in the failure of implantation.

  19. Connective tissue growth factor induces extracellular matrix in asthmatic airway smooth muscle

    NARCIS (Netherlands)

    Johnson, Peter R A; Burgess, Janette K; Ge, Qi; Poniris, Maree; Boustany, Sarah; Twigg, Stephen M; Black, Judith L

    2006-01-01

    Transforming growth factor (TGF)-beta and connective tissue growth factor may be implicated in extracellular matrix protein deposition in asthma. We have recently reported that TGF-beta increased connective tissue growth factor expression in airway smooth muscle cells isolated from patients with ast

  20. BIBF1120 inhibits fibroblasts proliferation and production of the extracellular matrix protein fibulin-1

    NARCIS (Netherlands)

    Burgess, Janette; Munk, Lizzie; Jaffar, Jade; Black, Judith; Oliver, Brian

    2015-01-01

    Introduction: In patients with idiopathic pulmonary fibrosis (IPF) transforming growth factorbeta 1 (TGFβ1) induces excessive extracellular matrix (ECM) protein deposition leading to fibrosis. Our recent studies have shown that the glycoprotein fibulin-1 is increased in serum and lung tissue from pa

  1. Rhinovirus infection induces extracellular matrix protein deposition in asthmatic and nonasthmatic airway smooth muscle cells

    NARCIS (Netherlands)

    Kuo, Curtis; Lim, Sam; King, Nicholas J C; Johnston, Sebastian L; Burgess, Janette K; Black, Judith L; Oliver, Brian G

    2011-01-01

    Airway remodeling, which includes increases in the extracellular matrix (ECM), is a characteristic feature of asthma and is correlated to disease severity. Rhinovirus (RV) infections are associated with increased risk of asthma development in young children and are the most common cause of asthma ex

  2. HEPARINS MODULATE EXTRACELLULAR-MATRIX AND PROTEIN-SYNTHESIS OF CULTURED RAT MESANGIAL CELLS

    NARCIS (Netherlands)

    WOLTHUIS, A; BOES, A; BERDEN, JHM

    Heparins blunt the development of glomerulosclerosis in several disease models in the rat and this protective effect may be related to suppression of glomerular cell proliferation. In this study the direct effect of heparins on another key event in glomerulosclerosis, extracellular matrix (ECM)

  3. Extracellular matrix formation enhances the ability of Streptococcus pneumoniae to cause invasive disease.

    Directory of Open Access Journals (Sweden)

    Claudia Trappetti

    Full Text Available 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.

  4. Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

    Science.gov (United States)

    Hermann, M; Jaconi, M E; Dahlgren, C; Waldvogel, F A; Stendahl, O; Lew, D P

    1990-09-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections.

  5. Local fluid transfer regulation in heart extracellular matrix.

    Science.gov (United States)

    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.

  6. [Effects of extracts of Dragon's blood on fibroblast proliferation and extracellular matrix hyaluronic acid].

    Science.gov (United States)

    Li, Dan; Hui, Rui; Hu, Yongwu; Han, Yan; Guo, Shuzhong

    2015-01-01

    To investigate the effects of Dragon' s blood extract on proliferation and secret extracellular matrix function of fibroblasts in vitro. Dragon' s blood was extracted by chloroform, acetoacetic ester, alcohol. Human fibroblast were cultured in vitro in media containing gradient dilutions of Dragon' s blood extracts (0.002, 0.02, 0.2, 2, 20 mg/ml) , which was followed by cell proliferation assessed with MTT assay on 0, 12, 24, 36, 48, 60, 72 h. Under the optimal concentration, the cell growth curves were drawn and the flow cytometry (FCM) was used to determine the changes of cell cycle. On 0, 12, 24, 36, 48, 60, 72 h, the concentration of hyaluronic acid in the supernatant of fibroblast culture was measured by radioimmunoassay. 0.2-2 mg/ml Dragon' s blood extracts enhanced the proliferation of fibroblasts in a dose-dependent manner. 2 mg/ml was the optimal dilution of Dragon's blood extract, and it increased the ratio of S cells in cell cycle [(25.80 ± 3.10)%] than control group [(7.50 ± 0.70)%, P Dragon's blood group, concentration of Hyaluronic acid secreted by fibroblasts gradually increased, but were less than control (P Dragon's blood acetoacetic ester extract improved the proliferation of cultured human fibroblasts in vitro, might be beneficial to promote wound healing.

  7. Linearized texture of three-dimensional extracellular matrix is mandatory for bladder cancer cell invasion

    Science.gov (United States)

    Alfano, Massimo; Nebuloni, Manuela; Allevi, Raffaele; Zerbi, Pietro; Longhi, Erika; Lucianò, Roberta; Locatelli, Irene; Pecoraro, Angela; Indrieri, Marco; Speziali, Chantal; Doglioni, Claudio; Milani, Paolo; Montorsi, Francesco; Salonia, Andrea

    2016-01-01

    In the fields of biomaterials and tissue engineering simulating the native microenvironment is of utmost importance. As a major component of the microenvironment, the extracellular matrix (ECM) contributes to tissue homeostasis, whereas modifications of native features are associated with pathological conditions. Furthermore, three-dimensional (3D) geometry is an important feature of synthetic scaffolds favoring cell stemness, maintenance and differentiation. We analyzed the 3D structure, geometrical measurements and anisotropy of the ECM isolated from (i) human bladder mucosa (basal lamina and lamina propria) and muscularis propria; and, (ii) bladder carcinoma (BC). Next, binding and invasion of bladder metastatic cell line was observed on synthetic scaffold recapitulating anisotropy of tumoral ECM, but not on scaffold with disorganized texture typical of non-neoplastic lamina propria. This study provided information regarding the ultrastructure and geometry of healthy human bladder and BC ECMs. Likewise, using synthetic scaffolds we identified linearization of the texture as a mandatory feature for BC cell invasion. Integrating microstructure and geometry with biochemical and mechanical factors could support the development of an innovative synthetic bladder substitute or a tumoral scaffold predictive of chemotherapy outcomes. PMID:27779205

  8. Interaction between extracellular matrix molecules and microbial pathogens: the missing link in autoimmunity?

    Directory of Open Access Journals (Sweden)

    Nidhi eSofat

    2015-01-01

    Full Text Available Rheumatoid arthritis (RA is an autoimmune disease characterised by inflammation, tissue rebuilding and fibrosis. Inability by the body to regulate inflammation effectively is one of the hallmarks of RA. Interactions between the external environment and the human host play an important role in the development of autoimmunity. In RA, the observation of anti-cyclic citrullinated peptide antibodies (ACPA to autoantigens is well recognised. Citrullination is a post-translational modification mediated by peptidyl arginine deiminases (PADs, which exist in both mammalian and bacterial forms. Previous studies have shown how proteins expressed in the human extracellular matrix (ECM acquire properties of damage-associated molecular patterns (DAMPs in RA and include collagens, tenascin-C and fibronectin. ECM DAMPs can further potentiate tissue damage in RA. Recent work has shown that citrullination in RA occurs at mucosal sites, including the oral cavity and lung. Mucosal sites have been linked with bacterial infection e.g. periodontal disease, where exogenous pathogens are implicated in the development of autoimmunity via an infectious trigger. Here we explore how mucosal surfaces exposed to bacteria could trigger autoimmunity in RA.

  9. Attachment of IgG to dermal extracellular matrix in patients with fibromyalgia.

    Science.gov (United States)

    Eneström, S; Bengtson, A; Lindström, F; Johan, K

    1990-01-01

    Deposits of IgG localized to collagen bundles/extracellular matrix components occurred in skin biopsies from patients with primary fibromyalgia (PF). None of these patients demonstrated a positive lupus band test. Control skin biopsies from healthy controls were negative but showed intense reactivity for IgG after collagenase treatment. PF-skin attached both homologous and heterologous serum IgG in indirect immunofluorescence, which may point to a qualitative alteration of dermal matrix components in PF. Skin from patients with systemic lupus erythematosus and rheumatoid arthritis showed a lower dermal fluorescence intensity than in PF patients. The cause of the presence of IgG in dermal tissue from PF patients is unclear. It may be caused by a non-specific attachment of IgG to the extracellular matrix related, for example, to tissue hypoxia and/or increased capillary leakage due to an increased number of mast cells in the PF-skin.

  10. Extracellular Matrix Remodeling of the Umbilical Cord in Pre-eclampsia as a Risk Factor for Fetal Hypertension

    Directory of Open Access Journals (Sweden)

    Lech Romanowicz

    2011-01-01

    Full Text Available The human umbilical cord forms a connection between the placenta and the foetus. It is composed of two arteries and one vein surrounded by Wharton's jelly. Pre-eclampsia is accompanied by extensive remodeling of extracellular matrix of umbilical cord. Matrix metalloproteinases (MMPs are engaged in degradation of extracellular matrix proteins and activation/inactivation of certain cytokines and enzymes. These enzymes will probably play a central role in the release of matrix-embedded cytokines and growth factors. MMP-2 (gelatinase A is the main collagenolytic enzyme of both umbilical artery and vein. Other metalloproteinases are present in several times lower amounts. Reduced activity of collagen-degrading enzymes may be a factor, which enhances the accumulation of collagen and some other proteins in the pre-eclamptic umbilical cord tissues. It seems to be possible that similar alterations occur in other fetal blood vessels. It may result in an increase in peripheral resistance as well as an increase in the blood pressure in the fetal vascular system. Some observations suggest that the raised pressure may persist after birth. Pre-eclampsia may be a factor that evokes an initiation of hypertension in utero and its amplification through childhood and adulthood.

  11. Biotensegrity of the extracellular matrix: physiology, dynamic mechanical balance and implications in oncology and mechanotherapy.

    Directory of Open Access Journals (Sweden)

    Irene eTadeo

    2014-03-01

    Full Text Available Cells have the capacity to convert mechanical stimuli into chemical changes. This process is based on the tensegrity principle, a mechanism of tensional integrity. To date, this principle has been demonstrated to act in physiological processes such as mechanotransduction and mechanosensing at different scales (from cell sensing through integrins to molecular mechanical interventions or even localized massage. The process involves intra- and extracellular components, including the participation of extracellular matrix and microtubules that act as compression structures, and actin filaments, which act as tension structures. The nucleus itself has its own tensegrity system which is implicated in cell proliferation, differentiation and apoptosis. Despite present advances, only the tip of the iceberg has so far been uncovered regarding the role of extracellular matrix compounds in influencing biotensegrity in pathological processes. Groups of cells, together with the surrounding ground substance, are subject to different and specific forces which certainly influence biological processes. In this paper we review the current knowledge on the role of extracellular matrix elements in determining biotensegrity in malignant processes, and describe their implication in therapeutic response, resistance to chemo- and radiotherapy, and subsequent tumor progression. Original data based on the study of neuroblastic tumors will be provided.

  12. Fractional Excretion of Survivin, Extracellular Matrix Metalloproteinase Inducer, and Matrix Metalloproteinase 7 in Children with Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    Agnieszka Bargenda

    2016-07-01

    Full Text Available Background: Epithelial–mesenchymal transition (EMT is defined as a transformation of tubular epithelial cells into mesenchymal ones. These cells migrate through the extracellular matrix and change into active myofibroblasts, which are responsible for excessive matrix deposition. Such changes may lead to tubular dysfunction and fibrosis of the renal parenchyma, characteristic of chronic kidney disease (CKD. However, there are no data on potential EMT markers in children with CKD. The aim of our study was to assess the usefulness of fractional excretion (FE of survivin, E-cadherin, extracellular matrix metalloproteinase inducer (EMMPRIN, matrix metalloproteinase (MMP7, and transforming growth factor beta 1 (TGF-β1 as potential markers of CKD-related complications such as tubular damage and fibrosis. Methods: Forty-one pre-dialysis children with CKD Stages 3–5 and 23 age-matched controls were enrolled in the study. The serum and urine concentrations of analysed parameters were assessed by an enzyme-linked immunosorbent assay test. Results: Tubular reabsorption of all analysed parameters was >99% in the control group. All FE values rose significantly in children with CKD, yet they remained 1%. Conclusions: FE of the examined markers may become a useful tool in the assessment of tubular dysfunction during the course of CKD. The FE of survivin, EMMPRIN, and MMP7 warrant further research as potential independent markers of kidney-specific EMT.

  13. Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion

    Science.gov (United States)

    He, Min; Storr-Paulsen, Thomas; Wang, Annie L.; Ghezzi, Chiara E.; Wang, Siran; Fullana, Matthew; Karamichos, Dimitrios; Utheim, Tor P.; Islam, Rakibul; Griffith, May; Islam, M. Mirazul; Hodges, Robin R.; Wnek, Gary E.; Kaplan, David L.; Dartt, Darlene A.

    2016-01-01

    Purpose We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. Methods We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Results Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Conclusions Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study. PMID:27832279

  14. Extracellular Matrix Stiffness Controls VEGF Signaling and Processing in Endothelial Cells.

    Science.gov (United States)

    Sack, Kelsey D; Teran, Madelane; Nugent, Matthew A

    2016-09-01

    Vascular endothelial growth factor A (VEGF) drives endothelial cell maintenance and angiogenesis. Endothelial cell behavior is altered by the stiffness of the substrate the cells are attached to suggesting that VEGF activity might be influenced by the mechanical cellular environment. We hypothesized that extracellular matrix (ECM) stiffness modifies VEGF-cell-matrix tethering leading to altered VEGF processing and signaling. We analyzed VEGF binding, internalization, and signaling as a function of substrate stiffness in endothelial cells cultured on fibronectin (Fn) linked polyacrylamide gels. Cell produced extracellular matrices on the softest substrates were least capable of binding VEGF, but the cells exhibited enhanced VEGF internalization and signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF and, inversely, heparin pre-treatment to enhance Fn-matrix binding of VEGF increased cell-internalization of VEGF regardless of matrix stiffness. β1 integrins, which connect cells to Fn, modulated VEGF uptake in a stiffness dependent fashion. Cells on hard surfaces showed decreased levels of activated β1 and inhibition of β1 integrin resulted in a greater proportional decrease in VEGF internalization than in cells on softer matrices. Extracellular matrix binding is necessary for VEGF internalization. Stiffness modifies the coordinated actions of VEGF-matrix binding and β1 integrin binding/activation, which together are critical for VEGF internalization. This study provides insight into how the microenvironment may influence tissue regeneration and response to injury and disease. J. Cell. Physiol. 231: 2026-2039, 2016. © 2016 Wiley Periodicals, Inc.

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

    Institute of Scientific and Technical Information of China (English)

    Diane M. Snow

    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 com-parisons 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, chon-droitin sulfate proteoglycans are the most widely researched and discussed proteoglycans in the ifeld of axonal injury and regen-eration. 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 speciifc chondroitin sulfate proteoglycan receptors and what this may mean for increased advancements in the ifeld (Shen), extracellular matrix degradation by matrix metallopro-teinases, which sculpt and resculpt to provide support for out-growth, 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.).

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

    Science.gov (United States)

    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.

  17. Phospholipids reduce gastric cancer cell adhesion to extracellular matrix in vitro

    Directory of Open Access Journals (Sweden)

    Jansen Petra

    2004-12-01

    Full Text Available Abstract Background Nidation of floating tumour cells initiates peritoneal carcinosis and limits prognosis of gastro-intestinal tumours. Adhesion of tumour cells to extracellular matrix components is a pivotal step in developing peritoneal dissemination of intraabdominal malignancies. Since phospholipids efficaciously prevented peritoneal adhesion formation in numerous animal studies we investigated their capacity to reduce adhesions of gastric cancer cells to extracellular matrix components (ECM. Methods Human gastric cancer cells (NUGC-4, Japanese Cancer Research Resources Bank, Tokyo, Japan were used in this study. Microtiter plates were coated with collagen IV (coll, laminin (ln and fibronectin (fn. Non-specific protein binding of the coated wells was blocked by adding 1% (w/v BSA (4°C, 12 h and rinsing the wells with Hepes buffer. 50.000 tumour cells in 100 μl medium were seeded into each well. Beside the controls, phospholipids were added in concentrations of 0.05, 0.1, 0.5, 0.75 and 1.0/100 μl medium. After an incubation interval of 30 min, attached cells were fixed and stained with 0.1% (w/v crystal violet. The dye was resuspended with 50 μl of 0.2% (v/v Triton X-100 per well and colour yields were then measured by an ELISA reader at 590 nm. Optical density (OD showed a linear relationship to the amount of cells and was corrected for dying of BSA/polystyrene without cells. Results The attachment of gastric cancer cells to collagen IV, laminin, and fibronectin could be significantly reduced up to 53% by phospholipid concentrations of 0.5 mg/100 μl and higher. Conclusion These results, within the scope of additional experimental studies on mice and rats which showed a significant reduction of peritoneal carcinosis, demonstrated the capacity of phospholipids in controlling abdominal nidation of tumour cells to ECM components. Lipid emulsions may be a beneficial adjunct in surgery of gastrointestinal malignancies.

  18. The Use of Natural Polymers in Tissue Engineering: A Focus on Electrospun Extracellular Matrix Analogues

    Directory of Open Access Journals (Sweden)

    Gary L. Bowlin

    2010-11-01

    Full Text Available Natural polymers such as collagens, elastin, and fibrinogen make up much of the body’s native extracellular matrix (ECM. This ECM provides structure and mechanical integrity to tissues, as well as communicating with the cellular components it supports to help facilitate and regulate daily cellular processes and wound healing. An ideal tissue engineering scaffold would not only replicate the structure of this ECM, but would also replicate the many functions that the ECM performs. In the past decade, the process of electrospinning has proven effective in creating non-woven ECM analogue scaffolds of micro to nanoscale diameter fibers from an array of synthetic and natural polymers. The ability of this fabrication technique to utilize the aforementioned natural polymers to create tissue engineering scaffolds has yielded promising results, both in vitro and in vivo, due in part to the enhanced bioactivity afforded by materials normally found within the human body. This review will present the process of electrospinning and describe the use of natural polymers in the creation of bioactive ECM analogues in tissue engineering.

  19. In vivo screening of extracellular matrix components produced under multiple experimental conditions implanted in one animal.

    Science.gov (United States)

    Higuera, Gustavo A; Hendriks, Jeanine A A; van Dalum, Joost; Wu, Ling; Schotel, Roka; Moreira-Teixeira, Liliana; van den Doel, Mirella; Leijten, Jeroen C H; Riesle, Jens; Karperien, Marcel; van Blitterswijk, Clemens A; Moroni, Lorenzo

    2013-06-01

    Animal experiments help to progress and ensure safety of an increasing number of novel therapies, drug development and chemicals. Unfortunately, these also lead to major ethical concerns, costs and limited experimental capacity. We foresee a coercion of all these issues by implantation of well systems directly into vertebrate animals. Here, we used rapid prototyping to create wells with biomaterials to create a three-dimensional (3D) well-system that can be used in vitro and in vivo. First, the well sizes and numbers were adjusted for 3D cell culture and in vitro screening of molecules. Then, the functionality of the wells was evaluated in vivo under 36 conditions for tissue regeneration involving human mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) screened in one animal. Each biocompatible well was controlled to contain μl-size volumes of tissue, which led to tissue penetration from the host and tissue formation under implanted conditions. We quantified both physically and biologically the amounts of extracellular matrix (ECM) components found in each well. Using this new concept the co-culture of hMSCs and bPCs was identified as a positive hit for cartilage tissue repair, which was a comparable result using conventional methods. The in vivo screening of candidate conditions opens an entirely new range of experimental possibilities, which significantly abates experimental animal use and increases the pace of discovery of medical treatments.

  20. Cadherin-11 is a novel regulator of extracellular matrix synthesis and tissue mechanics.

    Science.gov (United States)

    Row, Sindhu; Liu, Yayu; Alimperti, Stella; Agarwal, Sandeep K; Andreadis, Stelios T

    2016-08-01

    We discovered that Cadherin-11 (CDH11) regulates collagen and elastin synthesis, both affecting the mechanical properties and contractile function of animal tissues. Using a Cdh11-null mouse model, we observed a significant reduction in the mechanical properties [Youngs' modulus and ultimate tensile strength (UTS)] of Cdh11(-/-) as compared to wild-type (WT) mouse tissues, such as the aorta, bladder and skin. The deterioration of mechanical properties (Youngs' modulus and UTS) was accompanied by reduced collagen and elastin content in Cdh11(-/-) mouse tissues as well as in cells in culture. Similarly, knocking down CDH11 abolished collagen and elastin synthesis in human cells, and consequently reduced their ability to generate force. Conversely, engagement of CDH11 through homophilic interactions, led to swift activation of the TGF-β and ROCK pathways as evidenced by phosphorylation of downstream effectors. Subsequently, activation of the key transcription factors, MRTF-A (also known as MKL1) and MYOCD led to significant upregulation of collagen and elastin genes. Taken together, our results demonstrate a novel role of adherens junctions in regulating extracellular matrix (ECM) synthesis with implications for many important biological processes, including maintenance of tissue integrity, wound healing and tissue regeneration. © 2016. Published by The Company of Biologists Ltd.

  1. Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

    Directory of Open Access Journals (Sweden)

    David T. Ting

    2014-09-01

    Full Text Available Circulating tumor cells (CTCs are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

  2. Transchromosomic cell model of Down syndrome shows aberrant migration, adhesion and proteome response to extracellular matrix

    Directory of Open Access Journals (Sweden)

    Cotter Finbarr E

    2009-08-01

    Full Text Available Abstract Background Down syndrome (DS, caused by trisomy of human chromosome 21 (HSA21, is the most common genetic birth defect. Congenital heart defects (CHD are seen in 40% of DS children, and >50% of all atrioventricular canal defects in infancy are caused by trisomy 21, but the causative genes remain unknown. Results Here we show that aberrant adhesion and proliferation of DS cells can be reproduced using a transchromosomic model of DS (mouse fibroblasts bearing supernumerary HSA21. We also demonstrate a deacrease of cell migration in transchromosomic cells independently of their adhesion properties. We show that cell-autonomous proteome response to the presence of Collagen VI in extracellular matrix is strongly affected by trisomy 21. Conclusion This set of experiments establishes a new model system for genetic dissection of the specific HSA21 gene-overdose contributions to aberrant cell migration, adhesion, proliferation and specific proteome response to collagen VI, cellular phenotypes linked to the pathogenesis of CHD.

  3. Development of a porcine renal extracellular matrix scaffold as a platform for kidney regeneration.

    Science.gov (United States)

    Choi, Seock Hwan; Chun, So Young; Chae, Seon Yeong; Kim, Jin Rae; Oh, Se Heang; Chung, Sung Kwang; Lee, Jin Ho; Song, Phil Hyun; Choi, Gyu-Seog; Kim, Tae-Hwan; Kwon, Tae Gyun

    2015-04-01

    Acellular scaffolds, possessing an intact three-dimensional extracellular matrix (ECM) architecture and biochemical components, are promising for regeneration of complex organs, such as the kidney. We have successfully developed a porcine renal acellular scaffold and analyzed its physical/biochemical characteristics, biocompatibility, and kidney reconstructive potential. Segmented porcine kidney cortexes were treated with either 1% (v/v) Triton X-100 (Triton) or sodium dodecyl sulfate (SDS). Scanning electron microscopy showed both treatments preserved native tissue architecture, including porosity and composition. Swelling behavior was higher in the Triton-treated compared with the SDS-treated scaffold. Maximum compressive strength was lower in the Triton-treated compared with the SDS-treated scaffold. Attenuated total reflective-infrared spectroscopy showed the presence of amide II (-NH) in both scaffolds. Furthermore, richer ECM protein and growth factor contents were observed in the Triton-treated compared with SDS-treated scaffold. Primary human kidney cell adherence, viability, and proliferation were enhanced on the Triton-treated scaffold compared with SDS-treated scaffold. Following murine in vivo implantation, tumorigenecity was absent for both scaffolds after 8 weeks and in the Triton-treated scaffold only, glomeruli-like structure formation and neovascularity were observed. We identified 1% Triton X-100 as a more suitable decellularizing agent for porcine renal ECM scaffolds prior to kidney regeneration.

  4. Synthesis of hyaluronan haloacetates and biology of novel cross-linker-free synthetic extracellular matrix hydrogels.

    Science.gov (United States)

    Serban, Monica A; Prestwich, Glenn D

    2007-09-01

    Hyaluronan (HA) derivatives containing thiol-reactive electrophilic esters were prepared to react with thiol-modified macromolecules to give cross-linker-free hydrogels. Specifically, HA was converted to two haloacetate derivatives, HA bromoacetate (HABA) and HA iodoacetate (HAIA). In cytotoxicity assays, these reactive macromolecules predictably induced cell death in a dose-dependent manner. Cross-linker-free synthetic extracellular matrix (sECM) hydrogels were prepared by thiol alkylation using HAIA and HABA as polyvalent electrophiles and thiol-modified HA (CMHA-S) with or without thiol-modified gelatin (Gtn-DTPH) as polyvalent nucleophiles. When primary human fibroblasts were seeded on the surface of the sECMs containing only the electrophilic HA haloacetate and nucleophilic CMHA-S components, no significant cytoadherence was observed. Cell attachment and viability was 17% (HABA) to 30% (HAIA) lower on HA haloacetate cross-linked hydrogels than on CMHA-S that had been oxidatively cross-linked via disulfide-bonds. In contrast, sECMs that included Gtn-DTPH allowed fibroblasts to attach, spread, and proliferate. Taken together, the HA haloacetates are attractive candidates for producing cross-linker-free sECM biomaterials that can function either as anti-adhesive barriers or as cytoadhesive sECMs for cell culture in pseudo-3-D.

  5. Extracellular matrix proteins expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line.

    Science.gov (United States)

    Januchowski, Radosław; Zawierucha, Piotr; Ruciński, Marcin; Nowicki, Michał; Zabel, Maciej

    2014-01-01

    Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM) can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly--over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis.

  6. Role of Connective Tissue Growth Factor in Extracellular Matrix Degradation in Renal Tubular Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chun; ZHU Zhonghua; LIU Jianshe; YANG Xiao; FU Ling; DENG Anguo

    2007-01-01

    In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming growth factor β1 (TGF-β1) and to explore the role of CTGF in the degradation of renal extracellular matrix (ECM), a human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 (5 μg/L), the mRNA level of PAI-1 was detected by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may be a novel way in preventing renal fibrosis.

  7. Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

    Science.gov (United States)

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

    2014-01-01

    Non-typeable 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 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and 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 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were 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. PMID:24942343

  8. Nitric oxide regulates cell behavior on an interactive cell-derived extracellular matrix scaffold.

    Science.gov (United States)

    Xing, Qi; Zhang, Lijun; Redman, Travis; Qi, Shaohai; Zhao, Feng

    2015-12-01

    During tissue injury and wound healing process, there are dynamic reciprocal interactions among cells, extracellular matrix (ECM), and mediating molecules which are crucial for functional tissue repair. Nitric oxide (NO) is one of the key mediating molecules that can positively regulate various biological activities involved in wound healing. Various ECM components serve as binding sites for cells and mediating molecules, and the interactions further stimulate cellular activities. Human mesenchymal stem cells (hMSCs) can migrate to the wound site and contribute to tissue regeneration through differentiation and paracrine signaling. The objective of this work was to investigate the regulatory effect of NO on hMSCs in an interactive ECM-rich microenvironment. In order to mimic the in vivo stromal environment in wound site, a cell-derived ECM scaffold that was able to release NO within the range of in vivo wound fluid NO level was fabricated. Results showed that the micro-molar level of NO released from the ECM scaffold had an inhibitory effect on cellular activities of hMSCs. The NO impaired cell growth, altered cell morphology, disrupted the F-actin organization, also decreased the expression of focal adhesion related molecules integrin α5 and paxillin. These results may contribute to the elucidation of how NO acts on hMSCs in wound healing process.

  9. Extracellular Matrix-dependent Pathways in Colorectal Cancer Cell Lines Reveal Potential Targets for Anticancer Therapies.

    Science.gov (United States)

    Stankevicius, Vaidotas; Vasauskas, Gintautas; Noreikiene, Rimante; Kuodyte, Karolina; Valius, Mindaugas; Suziedelis, Kestutis

    2016-09-01

    Cancer cells grown in a 3D culture are more resistant to anticancer therapy treatment compared to those in a monolayer 2D culture. Emerging evidence has suggested that the key reasons for increased cell survival could be gene expression changes in cell-extracellular matrix (ECM) interaction-dependent manner. Global gene-expression changes were obtained in human colorectal carcinoma HT29 and DLD1 cell lines between 2D and laminin-rich (lr) ECM 3D growth conditions by gene-expression microarray analysis. The most significantly altered functional categories were revealed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The microarray data revealed that 841 and 1190 genes were differentially expressed in colorectal carcinoma DLD1 and HT29 cells. KEGG analysis indicated that the most significantly altered categories were cell adhesion, mitogen-activated protein kinase and immune response. Our results indicate altered pathways related to cancer development and progression and suggest potential ECM-regulated targets for the development of anticancer therapies. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  10. The alterations in the extracellular matrix composition guide the repair of damaged liver tissue.

    Science.gov (United States)

    Klaas, Mariliis; Kangur, Triin; Viil, Janeli; Mäemets-Allas, Kristina; Minajeva, Ave; Vadi, Krista; Antsov, Mikk; Lapidus, Natalia; Järvekülg, Martin; Jaks, Viljar

    2016-06-06

    While the cellular mechanisms of liver regeneration have been thoroughly studied, the role of extracellular matrix (ECM) in liver regeneration is still poorly understood. We utilized a proteomics-based approach to identify the shifts in ECM composition after CCl4 or DDC treatment and studied their effect on the proliferation of liver cells by combining biophysical and cell culture methods. We identified notable alterations in the ECM structural components (eg collagens I, IV, V, fibronectin, elastin) as well as in non-structural proteins (eg olfactomedin-4, thrombospondin-4, armadillo repeat-containing x-linked protein 2 (Armcx2)). Comparable alterations in ECM composition were seen in damaged human livers. The increase in collagen content and decrease in elastic fibers resulted in rearrangement and increased stiffness of damaged liver ECM. Interestingly, the alterations in ECM components were nonhomogenous and differed between periportal and pericentral areas and thus our experiments demonstrated the differential ability of selected ECM components to regulate the proliferation of hepatocytes and biliary cells. We define for the first time the alterations in the ECM composition of livers recovering from damage and present functional evidence for a coordinated ECM remodelling that ensures an efficient restoration of liver tissue.

  11. Extracellular Matrix Hydrogel Promotes Tissue Remodeling, Arteriogenesis, and Perfusion in a Rat Hindlimb Ischemia Model

    Directory of Open Access Journals (Sweden)

    Jessica L. Ungerleider, BS

    2016-01-01

    Full Text Available Although surgical and endovascular revascularization can be performed in peripheral arterial disease (PAD, 40% of patients with critical limb ischemia do not have a revascularization option. This study examines the efficacy and mechanisms of action of acellular extracellular matrix-based hydrogels as a potential novel therapy for treating PAD. We tested the efficacy of using a tissue-specific injectable hydrogel derived from decellularized porcine skeletal muscle (SKM and compared this to a new human umbilical cord-derived matrix (hUC hydrogel, which could have greater potential for tissue regeneration because of the younger age of the tissue source. In a rodent hindlimb ischemia model, both hydrogels were injected 1-week post-surgery and perfusion was regularly monitored with laser speckle contrast analysis to 35 days post-injection. There were significant improvements in hindlimb tissue perfusion and perfusion kinetics with both biomaterials. Histologic analysis indicated that the injected hydrogels were biocompatible, and resulted in arteriogenesis, rather than angiogenesis, as well as improved recruitment of skeletal muscle progenitors. Skeletal muscle fiber morphology analysis indicated that the muscle treated with the tissue-specific SKM hydrogel more closely matched healthy tissue morphology. Whole transcriptome analysis indicated that the SKM hydrogel caused a shift in the inflammatory response, decreased cell death, and increased blood vessel and muscle development. These results show the efficacy of an injectable ECM hydrogel alone as a potential therapy for treating patients with PAD. Our results indicate that the SKM hydrogel improved functional outcomes through stimulation of arteriogenesis and muscle progenitor cell recruitment.

  12. Genes responsible for vaginal extracellular matrix metabolism are modulated by women's reproductive cycle and menopause

    Directory of Open Access Journals (Sweden)

    Oksana Shynlova

    2013-04-01

    Full Text Available Objectives To analyze the expression of genes involved in extracellular matrix (ECM biogenesis and remodeling in vaginal tissue of women with clinically normal pelvic floor support (defined as controls according to the phase of menstrual cycle and postmenopausal women with and without pelvic organ prolapse (POP. Materials and Methods This study examined the expression of matrix metalloproteinases (MMPs, their tissue inhibitors (TIMPs, and the Lysyl oxidase (LOX family genes in the anterior vaginal wall of Caucasian women by real-time RT-PCR. Initially, mRNA expression was assessed in premenopausal controls in the secretory (group 1, n = 10 vs. proliferative (group 2, n = 8 phase of menstrual cycle. In addition, we compared premenopausal controls in the proliferative phase (group 2 vs. postmenopausal controls (group 3, n = 5. Finally, we analyzed postmenopausal controls (group 3 vs. postmenopausal women with advanced POP (group 4, n = 13. Results According to the phase of menstrual cycle, MMP1 was significantly reduced (p = 0.003, whereas the expression of TIMP1 and LOXL4 was significantly up-regulated during proliferative phase (both p < 0.01 when compared to the secretory phase in premenopausal control women. Regarding menopausal status/ageing, all MMPs were down-regulated, while TIMP3, TIMP4 and LOXL2 were significantly up-regulated in postmenopausal control women when compared to premenopausal controls (p = 0.005, p = 0.01 and p < 0.001, correspondingly. TIMP4 and LOXL2 mRNA levels were significantly decreased in postmenopausal POP patients compared to asymptomatic postmenopausal controls (p < 0.01 for both. Conclusions Our results indicate that ovarian cycle and age-related changes influence the expression of genes encoding proteins responsible for ECM metabolism in human vagina. Moreover, POP is associated with alteration in vaginal ECM components after menopause.

  13. Leptospira interrogans induces uterine inflammatory responses and abnormal expression of extracellular matrix proteins in dogs.

    Science.gov (United States)

    Wang, Wei; Gao, Xuejiao; Guo, Mengyao; Zhang, Wenlong; Song, Xiaojing; Wang, Tiancheng; Zhang, Zecai; Jiang, Haichao; Cao, Yongguo; Zhang, Naisheng

    2014-10-01

    Leptospira interrogans (L. interrogans), a worldwide zoonosis, infect humans and animals. In dogs, four syndromes caused by leptospirosis have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups), and also it caused inflammation. Extracellular matrix (ECM) is a complex mixture of matrix molecules that is crucial to the reproduction. Both inflammatory response and ECM are closed relative to reproductive. The aim of this study was to clarify how L. interrogans affected the uterus of dogs, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans. In the present study, 27 dogs were divided into 3 groups, intrauterine infusion with L. interrogans, to make uterine infection, sterile EMJH, and normal saline as a control, respectively. The uteruses were removed by surgical operation in 10, 20, and 30 days, respectively. The methods of histopathological analysis, ELISA, Western blot and qPCR were used. The results showed that L. interrogans induced significantly inflammatory responses, which were characterized by inflammatory cellular infiltration and high expression levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in uterine tissue of these dogs. Furthermore, L. interrogans strongly down-regulated the expression of ECM (collagens (CL) IV, fibronectins (FN) and laminins (LN)) in mRNA and protein levels. These data indicated that strongly inflammatory responses, and abnormal regulation of ECM might contribute to the proliferation of dogs infected by L. interrogans.

  14. The extracellular matrix of the lung and its role in edema formation

    Directory of Open Access Journals (Sweden)

    Paolo Pelosi

    2007-06-01

    Full Text Available The extracellular matrix is composed of a three-dimensional fiber mesh filled with different macromolecules such as: collagen (mainly type I and III, elastin, glycosaminoglycans, and proteoglycans. In the lung, the extracellular matrix has several functions which provide: 1 mechanical tensile and compressive strength and elasticity, 2 low mechanical tissue compliance contributing to the maintenance of normal interstitial fluid dynamics, 3 low resistive pathway for an effective gas exchange, d control of cell behavior by the binding of growth factors, chemokines, cytokines and the interaction with cell-surface receptors, and e tissue repair and remodeling. Fragmentation and disorganization of extracellular matrix components comprises the protective role of the extracellular matrix, leading to interstitial and eventually severe lung edema. Thus, once conditions of increased microvascular filtration are established, matrix remodeling proceeds fairly rapidly due to the activation of proteases. Conversely, a massive matrix deposition of collagen fiber decreases interstitial compliance and therefore makes the tissue safety factor stronger. As a result, changes in lung extracellular matrix significantly affect edema formation and distribution in the lung.A matriz extracelular é um aglomerado tridimensional demacromoléculas composta por: fibras colágenas (principalmente, tipos I e III, elastina, glicosaminoglicanos e proteoglicanos. No pulmão, a matriz extracelular tem várias funções, tais como: 1 promover estresse tensil e elasticidade tecidual, 2 contribuir para a manutenção da dinâmica de fluidos no interstício, 3 propiciar efetiva troca gasosa, 4 controlar a função celular através de sua ligação com fatores de crescimento, quimiocinas, citocinas e interação com receptores de superfície, e 5 remodelamento e reparo tecidual. A fragmentação e a desorganização da matriz extracelular pode acarretar edema intersticial e

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

    Science.gov (United States)

    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.

  16. Label-free Imaging of Arterial Cells and Extracellular Matrix Using a Multimodal CARS Microscope.

    Science.gov (United States)

    Wang, Han-Wei; Le, Thuc T; Cheng, Ji-Xin

    2008-04-01

    A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH(2)-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH(2) bonds. The extracellular matrix organization were further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

  17. How cells sense extracellular matrix stiffness: a material’s perspective

    Science.gov (United States)

    Trappmann, Britta; Chen, Christopher S.

    2014-01-01

    The mechanical properties of the extracellular matrix (ECM) in which cells reside have emerged as an important regulator of cell fate. While materials based on natural ECM have been used to implicate the role of substrate stiffness for cell fate decisions, it is difficult in these matrices to isolate mechanics from other structural parameters. In contrast, fully synthetic hydrogels offer independent control over physical and adhesive properties. New synthetic materials that also recreate the fibrous structural hierarchy of natural matrices are now being designed to study substrate mechanics in more complex ECMs. This perspective examines the ways in which new materials are being used to advance our understanding of how extracellular matrix stiffness impacts cell function. PMID:23611564

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

    Directory of Open Access Journals (Sweden)

    Livia Stavolone

    2017-09-01

    Full Text Available 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.

  19. Oral lichen sclerosus expressing extracellular matrix proteins and IgG4-positive plasma cells.

    Science.gov (United States)

    De Aquino Xavier, Flavia Calo; Prates, Alisio Alves; Gurgel, Clarissa Araujo; De Souza, Tulio Geraldo; Andrade, Rodrigo Guimaraes; Goncalves Ramos, Eduardo Antonio; Pedreira Ramalho, Luciana Maria; Dos Santos, Jean Nunes

    2014-09-16

    Lichen sclerosus (LS) is a mucocutaneous disease with uncommon oral involvement. The etiology is not yet well understood, but LS has been associated with autoimmune, genetic, and immunological factors. We report a 47-year-old man with LS that exhibited an asymptomatic white plaque with red patches on the maxillary alveolar mucosa extending to the labial mucosa. He had no other skin disease. Positive immunostaining for tenascin and scarcity of fibronectin suggested extracellular matrix reorganization. Elastin immunostaining indicated a reduction of elastic fibers. Immunoexpression of collagen IV in blood vessels and its absence in the epithelial basement membrane, together with diffuse MMP-9 immunoexpression, suggested altered proteolytic activity. Mast cell staining bordering areas of sclerosis indicated a possible role in the synthesis of collagen. IgG4 positivity in plasma cells suggested a role in the fibrogenesis. This is an unusual presentation of oral LS and we discuss immunohistochemical findings regarding cellular and extracellular matrix components.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  2. The ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes

    Directory of Open Access Journals (Sweden)

    Yue Gao

    2014-01-01

    Full Text Available Cartilage extracellular matrix (ECM is composed primarily of the network type II collagen (COLII and an interlocking mesh of fibrous proteins and proteoglycans (PGs, hyaluronic acid (HA, and chondroitin sulfate (CS. Articular cartilage ECM plays a crucial role in regulating chondrocyte metabolism and functions, such as organized cytoskeleton through integrin-mediated signaling via cell-matrix interaction. Cell signaling through integrins regulates several chondrocyte functions, including differentiation, metabolism, matrix remodeling, responses to mechanical stimulation, and cell survival. The major signaling pathways that regulate chondrogenesis have been identified as wnt signal, nitric oxide (NO signal, protein kinase C (PKC, and retinoic acid (RA signal. Integrins are a large family of molecules that are central regulators in multicellular biology. They orchestrate cell-cell and cell-matrix adhesive interactions from embryonic development to mature tissue function. In this review, we emphasize the signaling molecule effect and the biomechanics effect of cartilage ECM on chondrogenesis.

  3. Rat hair follicle dermal papillae have an extracellular matrix containing basement membrane components

    DEFF Research Database (Denmark)

    Couchman, J R

    1986-01-01

    Dermal papillae are small mesenchymally derived zones at the bases of hair follicles which have an important role in hair morphogenesis in the embryo and control of the hair growth cycle in postnatal mammals. The cells of the papilla are enmeshed in a dense extracellular matrix which undergoes...... extensive changes in concert with the hair cycle. Here it is shown that this matrix in anagen pelage follicles of postnatal rats contains an abundance of basement membrane components rather than dermal components such as interstitial collagens. In particular, type IV collagen, laminin, and basement membrane...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  6. Regulation of Non-Infectious Lung Injury, Inflammation, and Repair by the Extracellular Matrix Glycosaminoglycan Hyaluronan

    OpenAIRE

    Jiang, Dianhua; Liang, Jiurong; Noble, Paul W

    2010-01-01

    An important hallmark of tissue remodeling is the dynamic turnover of extracellular matrix (ECM). ECM performs a variety of functions in tissue repair including scaffold formation, modulation of fluid dynamics, and regulating cell behavior. During non-infectious tissue injury ECM degradation products are generated that acquire signaling functions not attributable to the native precursor molecules. Hyaluronan (HA) is a non-sulfated glycosaminoglycan which is produced in great abundance followi...

  7. Biomineralization of a Self-Assembled Extracellular Matrix for Bone Tissue Engineering

    OpenAIRE

    Meng, Yizhi; Qin, Yi-Xian; DiMasi, Elaine; Ba, Xiaolan; Rafailovich, Miriam; Pernodet, Nadine

    2008-01-01

    Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the understanding of bone formation and the development of a successfully engineered bone tissue scaffold. It is still unclear how ECM mechanical properties affect protein-mineral interactions in early stages of bone mineralization. We investigated the longitudinal mineralization properties of MC3T3-E1 cells and the elastic modulus of their ECM using shear modulation force microscopy, sync...

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ehrhart, E.J.; Gillette, E.L. [Colorado State Univ., Fort Collins, CO (United States); Barcellos-Hoff, M.H. [Univ. of California, Berkeley, CA (United States)

    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.

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

    Science.gov (United States)

    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 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 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.

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

    Science.gov (United States)

    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.

  12. Mouse basophils reside in extracellular matrix-enriched bone marrow niches which control their motility.

    Science.gov (United States)

    Smaniotto, Salete; Schneider, Elke; Goudin, Nicolas; Bricard-Rignault, Rachel; Machavoine, François; Dardenne, Mireille; Dy, Michel; Savino, Wilson

    2013-01-01

    Basophils co-express FcεRIα and CD49b, the α-2 chain of integrin-type receptor VLA-2 (α2β1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (α-4, α-5 and α-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow.

  13. Mouse basophils reside in extracellular matrix-enriched bone marrow niches which control their motility.

    Directory of Open Access Journals (Sweden)

    Salete Smaniotto

    Full Text Available Basophils co-express FcεRIα and CD49b, the α-2 chain of integrin-type receptor VLA-2 (α2β1, which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (α-4, α-5 and α-6 integrins subunits, respectively, we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow.

  14. Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects

    Energy Technology Data Exchange (ETDEWEB)

    Desai, Jayashree [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Shannon, Mark E. [Applied Biosystems; Johnson, Mahlon D. [University of Tennessee Graduate School of Medicine; Ruff, David W. [Applied Biosystems; Hughes, Lori A [ORNL; Kerley, Marilyn K [ORNL; Carpenter, D A [ORNL; Johnson, Dabney K [ORNL; Rinchik, Eugene M. [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Culiat, Cymbeline T [ORNL

    2006-01-01

    The mammalian Nell1 gene encodes a protein kinase C-b1 (PKC-b1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell16R, a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell16R has a T!A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell16R mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell16R mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

  15. Up-regulated expression of extracellular matrix remodeling genes in phagocytically challenged trabecular meshwork cells.

    Directory of Open Access Journals (Sweden)

    Kristine M Porter

    Full Text Available BACKGROUND: Cells in the trabecular meshwork (TM, the tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic function in TM cells is thought to play an important role in the normal functioning of the outflow pathway. Dysfunction of phagocytosis could lead to abnormalities of outflow resistance and increased intraocular pressure (IOP. However, the molecular mechanisms triggered by phagocytosis in TM cells are completely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profile analysis of human TM cells phagocytically challenged to E. coli or pigment under physiological and oxidative stress environment were performed using Affymetrix U133 plus 2.0 array and analyzed with Genespring GX. Despite the differential biological response elicited by E. coli and pigment particles, a number of genes, including MMP1, MMP3, TNFSF11, DIO2, KYNU, and KCCN2 showed differential expression with both phagocytic ligands in all conditions. Data was confirmed by qPCR in both human and porcine TM cells. Metacore pathway analysis and the usage of recombinant adenovirus encoding the dominant negative mutant of IkB identified NF-κB as a transcription factor mediating the up-regulation of at least MMP1 and MMP3 in TM cells with phagocytosis. In-gel zymography demonstrated increased collagenolytic and caseinolytic activities in the culture media of TM cells challenge to E. coli. In addition, collagenolytic I activity was further confirmed using the self-quenched fluorescent substrate DQ-Collagen I. CONCLUSIONS/SIGNIFICANCE: Here we report for the first time the differential gene expression profile of TM cells phagocytically challenged with either E. coli or pigment. Our data indicate a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

  16. Biomimetic aggrecan reduces cartilage extracellular matrix from degradation and lowers catabolic activity in ex vivo and in vivo models.

    Science.gov (United States)

    Sharma, Shaili; Lee, Aeju; Choi, Kuiwon; Kim, Kwangmeyung; Youn, Inchan; Trippel, Stephen B; Panitch, Alyssa

    2013-09-01

    Aggrecan, a major macromolecule in cartilage, protects the extracellular matrix (ECM) from degradation during the progression of osteoarthritis (OA). However, aggrecan itself is also susceptible to proteolytic cleavage. Here, the use of a biomimetic proteoglycan (mAGC) is presented, which functionally mimics aggrecan but lacks the known cleavage sites, protecting the molecule from proteolytic degradation. The objective of this study is to test the efficacy of this molecule in ex vivo (human OA synovial fluid) and in vivo (Sprague-Dawley rats) osteoarthritic models. These results indicate that mAGC's may protect articular cartilage against the loss of key ECM components, and lower catabolic protein and gene expression in both models. This suppression of matrix degradation has the potential to provide a healthy environment for tissue repair.

  17. [Findings in myocardial extracellular matrix in fatal poisoning with addictive substances].

    Science.gov (United States)

    Vajtr, D; Kukacka, J; Adámek, T

    2002-10-01

    It have been reported that chronic drug abuse induces changes in extracellular matrix (ECM) composition of myocardium. Matrix metaloproteinases (MMP) play important role in ECM remodelling and alteration in the ECM of myocardium. They are able to cleave ECM components and may contribute to progression of heart damage. Remodelling implies an alteration in the ECM and in the spatial orientation of cells and intercellular components. In this report we evaluated the extracellular matrix of myocardium of deceased drug abusers and controls. The myocardium of 5 toxicologically positive cases and 4 control cases were analysed by zymography of MMP, electrophoresis of collagens and histologicaly. Gelatinases (MMP-2 and MMP-9) and collagenase (MMP-1) activities were increased in drug abusers compare to the controls. In contrast, alpha chain of collagen type I after 160 hours collagen extraction, were not observed in control specimens. The activation of MMP proenzymes is one of the critical steps that lead to ECM breakdown. Our findings suggest that the myocardial collagen matrix is damaged in drug abusers.

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

    Directory of Open Access Journals (Sweden)

    Pollitt Christopher C

    2010-03-01

    Full Text Available Abstract Background 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. Results 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 (α6 and the hemidesmsome proteins, bullous pemphigoid antigen 1 (BP180 bullous pemphigoid antigen 2 (BP230 and plectin are also expressed. Conclusions 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.

  19. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

    Science.gov (United States)

    Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

    2016-02-17

    Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an

  20. The potential for targeting extracellular LOX proteins in human malignancy

    OpenAIRE

    Mayorca-Guiliani A; Erler JT

    2013-01-01

    Alejandro Mayorca-Guiliani, Janine T Erler Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Abstract: The extracellular matrix (ECM) is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX) enzymes cross-links collagens and elastin and, therefore, is a central player in ECM deposition and maturation. Exte...

  1. Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.

    Science.gov (United States)

    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

  2. Differential gene regulation under altered gravity conditions in follicular thyroid cancer cells: relationship between the extracellular matrix and the cytoskeleton

    NARCIS (Netherlands)

    Ulbrich, C.; Pietsch, J.; Grosse, J.; Wehland, M.; Schulz, H.; Saar, K.; Hübner, N.; Hauslage, J.; Hemmersbach, R.; Braun, M.; van Loon, J.; Vagt, N.; Egli, M.; Richter, P.; Einspanier, R.; Sharbati, S.; Baltz, T.; Infanger, M.; Ma, X.; Grimm, D.

    2011-01-01

    Extracellular matrix proteins, adhesion molecules, and cytoskeletal proteins form a dynamic network interacting with signalling molecules as an adaptive response to altered gravity. An important issue is the exact differentiation between real microgravity responses of the cells or cellular reactions

  3. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Tashiro, Kanae [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Shishido, Mayumi [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Fujimoto, Keiko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan); Hirota, Yuko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Yo, Kazuyuki; Gomi, Takamasa [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Tanaka, Yoshitaka, E-mail: tanakay@bioc.phar.kyushu-u.ac.jp [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan)

    2014-01-03

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

  4. Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease

    Directory of Open Access Journals (Sweden)

    Liu Bin

    2012-09-01

    Full Text Available Abstract Background Polycystic Kidney Disease (PKD kidneys exhibit increased extracellular matrix (ECM collagen expression and metalloproteinases (MMPs activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. Methods We examined the role of type I collagen (collagen I and membrane bound type 1 MMP (MT1-MMP on cyst development using both in vitro 3 dimensional (3D collagen gel culture and in vivo PCK rat model of PKD. Results We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Conclusions Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

  5. Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment.

    Science.gov (United States)

    Yonemura, Shigenobu

    2014-01-01

    Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.

  6. Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment.

    Directory of Open Access Journals (Sweden)

    Shigenobu Yonemura

    Full Text Available Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D culture systems rather than in two-dimensional (2-D culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules, EpH4 cells (mouse mammary gland, and R2/7 cells (human colon expressing wild-type α-catenin (R2/7 α-Cate cells. These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.

  7. Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy

    Directory of Open Access Journals (Sweden)

    Campagnola Paul J

    2010-03-01

    Full Text Available Abstract Background Remodeling of the extracellular matrix (ECM has been implicated in ovarian cancer, and we hypothesize that these alterations may provide a better optical marker of early disease than currently available imaging/screening methods and that understanding their physical manifestations will provide insight into invasion. Methods For this investigation we use Second Harmonic Generation (SHG imaging microcopy to study changes in the structure of the ovarian ECM in human normal and malignant ex vivo biopsies. This method directly visualizes the type I collagen in the ECM and provides quantitative metrics of the fibrillar assembly. To quantify these changes in collagen morphology we utilized an integrated approach combining 3D SHG imaging measurements and bulk optical parameter measurements in conjunction with Monte Carlo simulations of the experimental data to extract tissue structural properties. Results We find the SHG emission attributes (directionality and relative intensity and bulk optical parameters, both of which are related to the tissue structure, are significantly different in the tumors in a manner that is consistent with the change in collagen assembly. The normal and malignant tissues have highly different collagen fiber assemblies, where collectively, our findings show that the malignant ovaries are characterized by lower cell density, denser collagen, as well as higher regularity at both the fibril and fiber levels. This further suggests that the assembly in cancer may be comprised of newly synthesized collagen as opposed to modification of existing collagen. Conclusions Due to the large structural changes in tissue assembly and the SHG sensitivity to these collagen alterations, quantitative discrimination is achieved using small patient data sets. Ultimately these measurements may be developed as intrinsic biomarkers for use in clinical applications.

  8. Pentosan polysulfate decreases prostate smooth muscle proliferation and extracellular matrix turnover.

    Science.gov (United States)

    Elliot, S J; Zorn, B H; McLeod, D G; Moul, J W; Nyberg, L; Striker, L J; Striker, G E

    2003-01-01

    Benign prostatic hyperplasia (BPH) involves proliferation of smooth muscle cells and increased deposition of extracellular matrix (ECM). We recently found that pentosan polysulfate (PPS) has marked effects on growth and ECM of smooth muscle cells derived from vascular tissues. We examined smooth muscle cells cultured from human prostates and the effects of PPS on their growth and ECM production. Fragments of surgical prostatectomy specimens were diced, digested with collagenase (0.01%), and placed in culture medium supplemented with 20% fetal bovine serum. Outgrowths of elongated cells were characterized by light microscopic examination and immunohistochemical techniques by the presence of F-actin, alpha-smooth muscle actin, and myosin, which is a characteristic of smooth muscle cells. Two independent isolates were propagated, and growth curves and ECM production were assessed in the presence and absence of PPS (10 or 100 microg/ml). PPS decreased cell number beginning at day 1 and throughout the incubation period, up to 4 days. The amount of the ECM degradative enzymes, metallo-proteinases MMP-9 and MMP-2, was examined by zymography. PPS did not alter the amount of MMP-2 in the supernatants but MMP-9 was increased 234.4 +/- 17.23-fold over control cells. Tissue inhibitor of MMP (TIMPS), examined by reverse zymography, increased 200% over control. The amount of alpha I type (IV) and alpha I type (I) collagen released in the supernatant, measured by ELISA, significantly decreased in PPS-treated cultures. In conclusion, we found that the administration of PPS decreased proliferation as well as ECM production in prostate smooth muscle. Since smooth muscle proliferation and ECM are involved in the pathophysiology of BPH, PPS may have therapeutic potential.

  9. Podoplanin-mediated cell adhesion through extracellular matrix in oral squamous cell carcinoma.

    Science.gov (United States)

    Tsuneki, Masayuki; Yamazaki, Manabu; Maruyama, Satoshi; Cheng, Jun; Saku, Takashi

    2013-08-01

    Podoplanin (PDPN), one of the representative mucin-like type-I transmembrane glycoproteins specific to lymphatic endothelial cells, is expressed in various cancers including squamous cell carcinoma (SCC). On the basis of our previous studies, we have developed the hypothesis that PDPN functions in association with the extracellular matrix (ECM) from the cell surface side. The aim of this study was to elucidate the molecular role of PDPN in terms of cell adhesion, proliferation, and migration in oral SCC cells. Forty-four surgical specimens of oral SCC were used for immunohistochemistry for PDPN, and the expression profiles were correlated with their clinicopathological properties. Using ZK-1, a human oral SCC cell system, and five other cell systems, we examined PDPN expression levels by immunofluorescence, western blotting, and real-time PCR. The effects of transient PDPN knockdown by siRNA in ZK-1 were determined for cellular functions in terms of cell proliferation, adhesion, migration, and invasion in association with CD44 and hyaluronan. Cases without PDPN-positive cells were histopathologically classified as less-differentiated SCC, and SCC cells without PDPN more frequently invaded lymphatics. Adhesive properties of ZK-1 were significantly inhibited by siRNA, and PDPN was shown to collaborate with CD44 in cell adhesion to tether SCC cells with hyaluronan-rich ECM of the narrow intercellular space as well as with the stromal ECM. There was no siRNA effect in migration. We have demonstrated the primary function of PDPN in cell adhesion to ECM, which is to secondarily promote oral SCC cell proliferation.

  10. Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.

    Science.gov (United States)

    Pati, Falguni; Song, Tae-Ha; Rijal, Girdhari; Jang, Jinah; Kim, Sung Won; Cho, Dong-Woo

    2015-01-01

    3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals.

  11. Bioinspired Design of Polycaprolactone Composite Nanofibers as Artificial Bone Extracellular Matrix for Bone Regeneration Application.

    Science.gov (United States)

    Gao, Xiang; Song, Jinlin; Zhang, Yancong; Xu, Xiao; Zhang, Siqi; Ji, Ping; Wei, Shicheng

    2016-10-07

    The design and development of functional biomimetic systems for programmed stem cell response is a field of topical interest. To mimic bone extracellular matrix, we present an innovative strategy for constructing drug-loaded composite nanofibrous scaffolds in this study, which could integrate multiple cues from calcium phosphate mineral, bioactive molecule, and highly ordered fiber topography for the control of stem cell fate. Briefly, inspired by mussel adhesion mechanism, a polydopamine (pDA)-templated nanohydroxyapatite (tHA) was synthesized and then surface-functionalized with bone morphogenetic protein-7-derived peptides via catechol chemistry. Afterward, the resulting peptide-loaded tHA (tHA/pep) particles were blended with polycaprolactone (PCL) solution to fabricate electrospun hybrid nanofibers with random and aligned orientation. Our research demonstrated that the bioactivity of grafted peptides was retained in composite nanofibers. Compared to controls, PCL-tHA/pep composite nanofibers showed improved cytocompatibility. Moreover, the incorporated tHA/pep particles in nanofibers could further facilitate osteogenic differentiation potential of human mesenchymal stem cells (hMSCs). More importantly, the aligned PCL-tHA/pep composite nanofibers showed more osteogenic activity than did randomly oriented counterparts, even under nonosteoinductive conditions, indicating excellent performance of biomimetic design in cell fate decision. After in vivo implantation, the PCL-tHA/pep composite nanofibers with highly ordered structure could significantly promote the regeneration of lamellar-like bones in a rat calvarial critical-sized defect. Accordingly, the presented strategy in our work could be applied for a wide range of potential applications in not only bone regeneration application but also pharmaceutical science.

  12. Fate of distal lung epithelium cultured in a decellularized lung extracellular matrix.

    Science.gov (United States)

    Calle, Elizabeth A; Mendez, Julio J; Ghaedi, Mahboobe; Leiby, Katherine L; Bove, Peter F; Herzog, Erica L; Sundaram, Sumati; Niklason, Laura E

    2015-06-01

    Type II cells are the defenders of the alveolus. They produce surfactant to prevent alveolar collapse, they actively transport water to prevent filling of the air sacs that would otherwise prevent gas exchange, and they differentiate to type I epithelial cells. They are an indispensable component of functional lung tissue. To understand the functionality of type II cells in isolation, we sought to track their fate in decellularized matrices and to assess their ability to contribute to barrier function by differentiation to type I alveolar epithelial cells. Rat type II cells were isolated from neonatal rat lungs by labeling with the RTII-70 surface marker and separation using a magnetic column. This produced a population of ∼50% RTII-70-positive cells accompanied by few type I epithelial cells or α-actin-positive mesenchymal cells. This population was seeded into decellularized rat lung matrices and cultured for 1 or 7 days. Culture in Dulbecco's modified Eagle's medium +10% fetal bovine serum (FBS) resulted in reduced expression of epithelial markers and increased expression of mesenchymal markers. By 7 days, no epithelial markers were visible by immunostaining; nearly all cells were α-actin positive. Gene expression for the mesenchymal markers, α-actin, vimentin, and TGF-βR, was significantly upregulated on day 1 (p=0.0005, 0.0005, and 2.342E-5, respectively). Transcript levels of α-actin and TGF-βR remained high at 7 days (p=1.364E-10 and 0.0002). Interestingly, human type II cells cultured under the same conditions showed a similar trend in the loss of epithelial markers, but did not display high expression of mesenchymal markers. Rat cells additionally showed the ability to produce and degrade the basement membrane and extracellular matrix components, such as fibronectin, collagen IV, and collagen I. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) showed significant increases in expression of the fibronectin and matrix

  13. Lib, transcriptionally induced in senile plaque-associated astrocytes, promotes glial migration through extracellular matrix.

    Science.gov (United States)

    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.

  14. Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine

    Directory of Open Access Journals (Sweden)

    Mikaël M Martino

    2015-04-01

    Full Text Available Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localization of angiogenic signals in the extracellular matrix is crucial to ensure the proper assembly and maturation of new vascular structures. Here we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of extracellular matrix to optimize the signaling microenvironment of vascular growth factors.

  15. Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Xiao-Ping Chen; Wan-Guang Zhang; Feng Zhang; Shuai Xiang; Han-Hua Dong; Lei Zhang

    2009-01-01

    AIM: To elucidate the interaction between nonparenchymal 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.

  16. Excess genistein suppresses the synthesis of extracellular matrix in female rat mandibular condylar cartilage

    Institute of Scientific and Technical Information of China (English)

    Shi-bin YU; Xiang-hui XING; Guang-ying DONG; Xi-li WENG; Mei-qing WANG

    2012-01-01

    Aim:To investigate the effect of excess genistein on the extracellular matrix in mandibular condylar cartilage of female rats in vivo.Methods:Female SD rats were administered through oral gavage with genistein (50 mg/kg) or placebo daily for 6 weeks.The morphological changes of temporomandibular joints were studied with HE staining.The expression of cartilage matrix compounds (aggrecan and collagen type Ⅱ),estrogen-related molecules (aromatase,estradiol,ERα and ERβ) and proliferating cell nuclear antigen (PCNA) in mandibular condylar cartilage was detected using immunohistochemistry,ELISA and real-time PCR.Results:The genistein treatment significantly reduced the thickness of the posterior and middle regions of mandibular condylar cartilage,and decreased the expression of collagen type Ⅱ,aggrecan and PCNA.Compared with the control group,the estradiol content and expression levels of the key estradiol-synthesizing enzyme aromatase in the genistein-treatment group were significantly decreased.The genistein treatment significantly increased the expression of ERβ,but decreased the expression of ERα.Conclusion:Excess genistein suppresses extracellular matrix synthesis and chondrocytes proliferation,resulting in thinner mandibular condylar cartilage.These effects may be detrimental to the ability of mandibular condylar cartilage to adapt to mechanical loads.

  17. Decellularization of porcine skeletal muscle extracellular matrix for the formulation of a matrix hydrogel: a preliminary study.

    Science.gov (United States)

    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 collagen and glycosaminoglycan, with a total protein concentration of 64.8 ± 6.9%. An acellular ECM hydrogel from porcine skeletal muscle was efficiently produced.

  18. EcmPred: Prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection

    KAUST Repository

    Kandaswamy, Krishna Kumar Umar

    2013-01-01

    The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan syndrome, osteogenesis imperfecta, numerous chondrodysplasias, and skin diseases. In this work, we report a random forest approach, EcmPred, for the prediction of ECM proteins from protein sequences. EcmPred was trained on a dataset containing 300 ECM and 300 non-ECM and tested on a dataset containing 145 ECM and 4187 non-ECM proteins. EcmPred achieved 83% accuracy on the training and 77% on the test dataset. EcmPred predicted 15 out of 20 experimentally verified ECM proteins. By scanning the entire human proteome, we predicted novel ECM proteins validated with gene ontology and InterPro. The dataset and standalone version of the EcmPred software is available at http://www.inb.uni-luebeck.de/tools-demos/Extracellular_matrix_proteins/EcmPred. © 2012 Elsevier Ltd.

  19. Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc

    Directory of Open Access Journals (Sweden)

    Mwale F.

    2004-12-01

    Full Text Available Tissue engineering of intervertebral discs (IVD using mesenchymal stem cells (MSCs induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. However, since there is no unique marker characteristic of discs and since hyaline cartilage and immature nucleus pulposus (NP possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. This study was performed to compare the proteoglycan to collagen ratio (measured as GAG to hydroxyproline ratio in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals and test the hypothesis that this ratio can be used for in vivo studies to distinguish between a normal NP and hyaline cartilage phenotype. Whole human lumbar spine specimens from fresh cadavers, ranging in age from 12 weeks to 79 years, were used to harvest the IVDs and adjacent endplates. The GAG to hydroxyproline ratio within the NP of young adults is approximately 27:1, whereas the ratio within the hyaline cartilage endplate of the same aged individuals is about 2:1. The production of an extracellular matrix with a high proteoglycan to collagen ratio can be used in vivo to distinguish NP cells from chondrocytes, and could help in identifying a NP-like phenotype in vivo as opposed to a chondrocyte when MSCs are induced to differentiate for tissue engineering of a disc.

  20. EcmPred: prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection.

    Science.gov (United States)

    Kandaswamy, Krishna Kumar; Pugalenthi, Ganesan; Kalies, Kai-Uwe; Hartmann, Enno; Martinetz, Thomas

    2013-01-21

    The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan syndrome, osteogenesis imperfecta, numerous chondrodysplasias, and skin diseases. In this work, we report a random forest approach, EcmPred, for the prediction of ECM proteins from protein sequences. EcmPred was trained on a dataset containing 300 ECM and 300 non-ECM and tested on a dataset containing 145 ECM and 4187 non-ECM proteins. EcmPred achieved 83% accuracy on the training and 77% on the test dataset. EcmPred predicted 15 out of 20 experimentally verified ECM proteins. By scanning the entire human proteome, we predicted novel ECM proteins validated with gene ontology and InterPro. The dataset and standalone version of the EcmPred software is available at http://www.inb.uni-luebeck.de/tools-demos/Extracellular_matrix_proteins/EcmPred.

  1. Inflammation and breast cancer. Metalloproteinases as common effectors of inflammation and extracellular matrix breakdown in breast cancer

    Science.gov (United States)

    Hojilla, Carlo V; Wood, Geoffrey A; Khokha, Rama

    2008-01-01

    Two rapidly evolving fields are converging to impact breast cancer: one has identified novel substrates of metalloproteinases that alter immune cell function, and the other has revealed a role for inflammation in human cancers. Evidence now shows that the mechanisms underlying these two fields interact in the context of breast cancer, providing new opportunities to understand this disease and uncover novel therapeutic strategies. The metalloproteinase class of enzymes is well studied in mammary gland development and physiology, but mostly in the context of extracellular matrix modification. Aberrant metalloproteinase expression has also been implicated in breast cancer progression, where these genes act as tumor modifiers. Here, we review how the metalloproteinase axis impacts mammary physiology and tumorigenesis and is associated with inflammatory cell influx in human breast cancer, and evaluate its potential as a regulator of inflammation in the mammary gland. PMID:18394187

  2. Contribution of the extracellular matrix to the viscoelastic behavior of the urinary bladder wall.

    Science.gov (United States)

    Nagatomi, Jiro; Toosi, Kevin K; Chancellor, Michael B; Sacks, Michael S

    2008-10-01

    We previously reported that when the stress relaxation response of urinary bladder wall (UBW) tissue was analyzed using a single continuous reduced relaxation function (RRF), we observed non-uniformly distributed, time-dependent residuals (Ann Biomed Eng 32(10):1409-1419, 2004). We concluded that the single relaxation spectrum was inadequate and that a new viscoelastic model for bladder wall was necessary. In the present study, we report a new approach composed of independent RRFs for smooth muscle and the extracellular matrix components (ECM), connected through a stress-dependent recruitment function. In order to determine the RRF for the ECM component, biaxial stress relaxation experiments were first performed on decellularized extracellular matrix network of the bladder obtained from normal and spinal cord injured rats. While it was assumed that smooth muscle followed a single spectrum RRF, modeling the UBW ECM required a dual-Gaussian spectrum. Experimental results revealed that the ECM stress relaxation response was insensitive to the initial stress level. Thus, the average ECM RRF parameters were determined by fitting the average stress relaxation data. The resulting stress relaxation behavior of whole bladder tissue was modeled by combining the ECM RRF with the RRF for the smooth muscle component using an exponential recruitment function representing the recruitment of collagen fibers at higher stress levels. In summary, the present study demonstrated, for the first time, that stress relaxation response of bladder tissue can be better modeled when divided into the contributions of the extracellular matrix and smooth muscle components. This modeling approach is suitable for prediction of mechanical behaviors of the urinary bladder and other organs that exhibit rapid tissue remodeling (i.e., smooth muscle hypertrophy and altered ECM synthesis) under various pathological conditions.

  3. Extracellular matrix assembly in extreme acidic eukaryotic biofilms and their possible implications in heavy metal adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Aguilera, Angeles [Centro de Astrobiologia (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain)], E-mail: aguileraba@inta.es; Souza-Egipsy, Virginia [Centro de Astrobiologia (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain); San Martin-Uriz, Patxi [Centro de Biologia Molecular (UAM-CSIC), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Amils, Ricardo [Centro de Astrobiologia (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain); Centro de Biologia Molecular (UAM-CSIC), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

    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 Rio 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{sup -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{sup -1} dry weight; 10% to 30% of the total biofilm dry weight. Capsular EPS ranged from 50 to 318 mg g{sup -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{sup -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.

  4. Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold.

    Science.gov (United States)

    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.

  5. Dextran sulphate crowding and sodium deoxycholate lysis of primary breast fibroblast cells achieve extracellular matrix deposition and decellularization for breast cancer stem cell culture

    Directory of Open Access Journals (Sweden)

    Aroem Naruni

    2016-01-01

    Full Text Available AbstrakLatar belakang: Lingkungan mikro yaitu sel stromal dam matriks ekstraseluler saat ini dinyatakansebagai kontributor dalam perkembangan tumor. Beberapa penelitian telah mengembangkan matriksekstraseluler yang mendukung perkembangan sel in vitro. Matriks ekstraseluler adalah suatu komplekssusunan supramolekuler dari berbagai macam glycoprotein dan proteoglycan. Matriks ekstraselulermenyediakan integritas jaringan, bertindak sebagai scaffold alami tempat sel melekat dan berinteraksiserta berperan sebagai reservoir pertumbuhan sel. Penelitian ini bertujuan untuk mendapatkan deposisidan deselularisasi yang optimal pada matriks ekstraseluler.Metode: Dalam penelitian ini, kami mengembangkan cells crowder untuk meningkatkan deposit matriksekstraseluler dari kultur sel primer fibroblast payudara yang diperoleh dari spesimen hasil operasimammoplasty. Dextran 500 kDa ditambahkan dalam media kultur DMEM lengkap yang telah ditambahkan0.5% FBS dan 100μM L-ascorbic acid 2-phosphate. Setelah tujuh hari, sel dilisis dengan menggunakanSodium Deoxycolate (DOC.Hasil: Deposisi matriks ekstraseluler dan proses deselulerisasi dari sel primer fibroblas payudara dapatterdeteksi dengan menggunakan antibodi Rabbit anti human fibronectin yang selanjutnya ditambahkandengan anti rabbit IgG yang telah dikonjugasi dengan Alexa Fluor 488.Kesimpulan: Penambahan dextran sulfat dan prosesing lysis dengan sodium deoxycolate dapatmeningkatkan deposisi dan menghasilkan deselularisasi matriks ekstraseluler. (Health Science Journalof Indonesia 2015;6:43-7Kata kunci: matriks ekstra selular, kanker mammae, stem cell, sel fibroblast AbstractBackground: The microenvironment including stromal cells and extracellular matrix (ECM is now consideredan active contributor to tumor progression. Certain studies have developed ECM which supports a suitable cellulargrowth in vitro. The ECM is a complex supramolecular assembly of a variety of glycoproteins and proteoglycans.Extracellular

  6. In vitro enhancement of extracellular matrix formation as natural bioscaffold for stem cell culture

    Science.gov (United States)

    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.

  7. Mouse Basophils Reside in Extracellular Matrix-Enriched Bone Marrow Niches Which Control Their Motility

    OpenAIRE

    Salete Smaniotto; Elke Schneider; Nicolas Goudin; Rachel Bricard-Rignault; François Machavoine; Mireille Dardenne; Michel Dy; Wilson Savino

    2013-01-01

    Basophils co-express FcεRIα and CD49b, the α-2 chain of integrin-type receptor VLA-2 (α2β1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (...

  8. Adhesion molecules and the extracellular matrix as drug targets for glioma.

    Science.gov (United States)

    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.

  9. Of extracellular matrix, scaffolds, and signaling: Tissuearchitectureregulates development, homeostasis, and cancer

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Extracellular matrix molecules play diverse roles in the growth and guidance of central nervous system axons

    Directory of Open Access Journals (Sweden)

    M.A. Pires-Neto

    1999-05-01

    Full Text Available Axon growth and guidance represent complex biological processes in which probably intervene diverse sets of molecular cues that allow for the appropriate wiring of the central nervous system (CNS. The extracellular matrix (ECM represents a major contributor of molecular signals either diffusible or membrane-bound that may regulate different stages of neural development. Some of the brain ECM molecules form tridimensional structures (tunnels and boundaries that appear during time- and space-regulated events, possibly playing relevant roles in the control of axon elongation and pathfinding. This short review focuses mainly on the recognized roles played by proteoglycans, laminin, fibronectin and tenascin in axonal development during ontogenesis.

  11. Extracellular Protein Interactions Mediated by the Neural Cell Adhesion Molecule, NCAM: Heterophilic Interactions Between NCAM and Cell Adhesion Molecules, Extracellular Matrix Proteins, and Viruses

    DEFF Research Database (Denmark)

    Nielsen, Janne; Kulahin, Nikolaj; Walmod, Peter

    2008-01-01

    Cell adhesion molecules (CAMs) mediate cell-to-cell interactions and interactions between cells and the extracellular matrix (ECM). The neural cell adhesion molecule (NCAM), a prototypic member of the immunoglobulin (Ig) superfamily of CAMs, mediates adhesion through homophilic and heterophilic i...

  12. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium

    Science.gov (United States)

    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.

  13. Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix

    Science.gov (United States)

    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

  14. Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells

    Science.gov (United States)

    Chen, Qike K.; Lee, KangAe; Radisky, Derek C.; Nelson, Celeste M.

    2013-01-01

    Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland. PMID:23660532

  15. Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins.

    Science.gov (United States)

    Clough, Bret H; McCarley, Matthew R; Krause, Ulf; Zeitouni, Suzanne; Froese, Jeremiah J; McNeill, Eoin P; Chaput, Christopher D; Sampson, H Wayne; Gregory, Carl A

    2015-01-01

    Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.

  16. Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix.

    Science.gov (United States)

    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.

  17. Extra-cellular matrix changes in Schistosoma mansoni-infected Biomphalaria glabrata

    Directory of Open Access Journals (Sweden)

    Borges Claudia Maria da Cunha

    2003-01-01

    Full Text Available Reactivity of snails against parasites exhibits a primitive focal reaction, with encapsulation, phagocytosis and destruction of parasite larvae by macrophage-like cells - the hemocytes. This reaction mimics granulomatous inflammation seen in higher animals. However, different from the latter, little is known about the participation of extra-cellular matrix in such snail defense reactions. Normal and Schistosoma mansoni-infected Biomphalaria glabrata of different strains were submitted to cytological, histological, ultrastructural and biochemical methods in order to investigate the behavior of extra-cellular tissues at the site of anti-parasite reactions. In spite of the presence of two cell-types in peripheral hemolymph, only one cell-type was present at the sites of tissue reactions. Although pre-existent collagen and elastic fibers and microfibrils sometimes appeared slightly compressed around focal reactions, no evidences of duplication, synthesis or deposition of connective-tissue extra-cellular components were observed within or around the zones of reactive cell accumulations. Thus, tissue reactions against S. mansoni in the snail B. glabrata appeared exclusively dependent on one specific population of hemocytes.

  18. Right ventricular function after repair of tetralogy of Fallot: a comparison between bovine pericardium and porcine small intestinal extracellular matrix.

    Science.gov (United States)

    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.

  19. Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix

    Directory of Open Access Journals (Sweden)

    Ramírez G.

    1999-01-01

    Full Text Available As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.

  20. Bubaline Cholecyst Derived Extracellular Matrix for Reconstruction of Full Thickness Skin Wounds in Rats

    Directory of Open Access Journals (Sweden)

    Poonam Shakya

    2016-01-01

    Full Text Available 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.

  1. Sesamin inhibits lipopolysaccharide-induced inflammation and extracellular matrix catabolism in rat intervertebral disc.

    Science.gov (United States)

    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.

  2. Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms.

    Science.gov (United States)

    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.

  3. In the presence of danger:the extracellular matrix defensive response to central nervous system injury

    Institute of Scientific and Technical Information of China (English)

    Lyn B. Jakeman; Kent E. Williams; Bryan Brautigam

    2014-01-01

    Glial cells in the central nervous system (CNS) contribute to formation of the extracellular matrix, which provides adhesive sites, signaling molecules, and a diffusion barrier to enhance efifcient neurotransmission and axon potential propagation. In the normal adult CNS, the extracellular matrix (ECM) is relatively stable except in selected regions characterized by dynamic remodel-ing. However, after trauma such as a spinal cord injury or cortical contusion, the lesion epicenter becomes a focus of acute neuroinlfammation. The activation of the surrounding glial cells leads to a dramatic change in the composition of the ECM at the edges of the lesion, creating a perile-sion environment dominated by growth inhibitory molecules and restoration of the peripheral/central nervous system border. An advantage of this response is to limit the invasion of damaging cells and diffusion of toxic molecules into the spared tissue regions, but this occurs at the cost of inhibiting migration of endogenous repair cells and preventing axonal regrowth. The following review was prepared by reading and discussing over 200 research articles in the ifeld published in PubMed and selecting those with signiifcant impact and/or controversial points. This article highlights structural and functional features of the normal adult CNS ECM and then focuses on the reactions of glial cells and changes in the perilesion border that occur following spinal cord or contusive brain injury. Current research strategies directed at modifying the inhibitory perile-sion microenvironment without eliminating the protective functions of glial cell activation are discussed.

  4. Regulation of PDGFC signalling and extracellular matrix composition by FREM1 in mice

    Directory of Open Access Journals (Sweden)

    Fenny Wiradjaja

    2013-11-01

    Fras1-related extracellular matrix protein 1 (FREM1 is required for epidermal adhesion during embryogenesis, and mice lacking the gene develop fetal skin blisters and a range of other developmental defects. Mutations in members of the FRAS/FREM gene family cause diseases of the Fraser syndrome spectrum. Embryonic epidermal blistering is also observed in mice lacking PdgfC and its receptor, PDGFRα. In this article, we show that FREM1 binds to PDGFC and that this interaction regulates signalling downstream of PDGFRα. Fibroblasts from Frem1-mutant mice respond to PDGFC stimulation, but with a shorter duration and amplitude than do wild-type cells. Significantly, PDGFC-stimulated expression of the metalloproteinase inhibitor Timp1 is reduced in cells with Frem1 mutations, leading to reduced basement membrane collagen I deposition. These results show that the physical interaction of FREM1 with PDGFC can regulate remodelling of the extracellular matrix downstream of PDGFRα. We propose that loss of FREM1 function promotes epidermal blistering in Fraser syndrome as a consequence of reduced PDGFC activity, in addition to its stabilising role in the basement membrane.

  5. Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro.

    Science.gov (United States)

    Alibardi, Lorenzo; Polazzi, Elisabetta

    2012-04-01

    The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.

  6. The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

    Science.gov (United States)

    Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

    2011-10-01

    Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.

  7. Novel insights into the function and dynamics of extracellular matrix in liver fibrosis

    DEFF Research Database (Denmark)

    Karsdal, Morten A; Manon-Jensen, Tina; Genovese, Federica

    2015-01-01

    Emerging evidence suggests that altered components and posttranslational modifications of proteins in the extracellular matrix (ECM) may both initiate and drive disease progression. The ECM is a complex grid consisting of multiple proteins, most of which play a vital role in containing the essent......Emerging evidence suggests that altered components and posttranslational modifications of proteins in the extracellular matrix (ECM) may both initiate and drive disease progression. The ECM is a complex grid consisting of multiple proteins, most of which play a vital role in containing......) explore key structural and functional components of the ECM as exemplified by monogenetic disorders leading to severe pathologies, 2) discuss selected pathological posttranslational modifications of ECM proteins resulting in altered functional (signaling) properties from the original structural proteins......, and 3) discuss how these findings support the novel concept that an increasing number of components of the ECM harbor signaling functions that can modulate fibrotic liver disease. The ECM entails functions in addition to anchoring cells and modulating their migratory behavior. Key ECM components...

  8. Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

    Science.gov (United States)

    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.

  9. Expression of extracellular matrix proteins: tenascin-C, fibronectin and galectin-3 in prostatic adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Monika Ulamec

    2015-12-01

    Full Text Available Introduction: The interchanged stromal-epithelial relations and altered expression profiles of various extracellular matrix (ECM proteins creates a suitable microenvironment for cancer development and growth. We support the opinion that remodeling of the extracellular matrix (ECM plays an important role in the cancer progression. The aim of this study was to examine the expression of ECM proteins tenascin-C, fibronectin and galectin-3 in prostatic adenocarcinoma. Methods: Glands and surrounding stroma were analyzed in randomly selected specimens from 52 patients with prostate cancer and 28 patients with benign prostatic hyperplasia (BHP. To evaluate the intensity of tenascin-C, fibronectin and galectin-3 expression the percentage of positively immunostained stromal cells was examined.Results: Compared to BPH, stroma of prostatic adenocarcinoma showed statistically significant increase in tenascin-C expression (p<0.001, predominantly around neoplastic glands, while fibronectin (p=0.001 and galectin-3 (p<0.001 expression in the same area was decreased.Conclusions: Our study confirms changes in the expression of ECM proteins of prostate cancer which may have important role in the cancer development.

  10. Up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver.

    Science.gov (United States)

    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.

  11. Salvage of Failed Local and Regional Flaps with Porcine Urinary Bladder Extracellular Matrix Aided Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    Gregory J. Kruper

    2013-01-01

    Full Text Available Local and regional flap failure can be a major complication in head and neck surgery, which continue to be prevalent for a number of reasons including poor flap design, improper surgical technique, and poor tissue vascularity. Dealing with these failures can be quite difficult. Surgical debridement, flap revisions, and complex wound regimens are necessitated to reestablish appropriate tissue coverage. Traditional use of wet to dry dressing to enable proper wound granulation and possible closure with additional flaps or skin grafts is a laborious process. Such treatments place great time burdens on the patient, physicians, and nurses. Because the face and neck possess a complex three-dimensional topography, wound dressings are inherently complex to design and change. Many patients also require postoperative treatments such as radiation and chemotherapy to treat aggressive malignancies, and delay in wound healing leads to a delay in adjuvant treatment. Recently, advances in regenerative medicine, specifically xenogeneic extracellular matrix compounds, have been shown to promote tissue growth while limiting scar tissue formation (Badylak 2004. To our knowledge, this paper is the first case series using the porcine extracellular matrix bioscaffold (MatriStem ACell, Columbia, MD, USA to salvage flaps with extensive wound breakdown on the face and neck.

  12. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

    Directory of Open Access Journals (Sweden)

    Hosoe Misa

    2010-06-01

    Full Text Available Abstract Background Extracellular matrix metalloproteinase inducer (EMMPRIN regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow. Methods In this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis. Results EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2 and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium. Conclusion EMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14

  13. Ubiquitylation functions in the calcium carbonate biomineralization in the extracellular matrix.

    Directory of Open Access Journals (Sweden)

    Dong Fang

    Full Text Available 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.

  14. Naturally Occurring Extracellular Matrix Scaffolds for Dermal Regeneration: Do They Really Need Cells?

    Directory of Open Access Journals (Sweden)

    A. M. Eweida

    2015-01-01

    Full Text Available The pronounced effect of extracellular matrix (ECM scaffolds in supporting tissue regeneration is related mainly to their maintained 3D structure and their bioactive components. These decellularized matrix scaffolds could be revitalized before grafting via adding stem cells, fibroblasts, or keratinocytes to promote wound healing. We reviewed the online published literature in the last five years for the studies that performed ECM revitalization and discussed the results of these studies and the related literature. Eighteen articles met the search criteria. Twelve studies included adding cells to acellular dermal matrix (ADM, 3 studies were on small intestinal mucosa (SIS, one study was on urinary bladder matrix (UBM, one study was on amniotic membrane, and one study included both SIS and ADM loaded constructs. We believe that, in chronic and difficult-to-heal wounds, revitalizing the ECM scaffolds would be beneficial to overcome the defective host tissue interaction. This belief still has to be verified by high quality randomised clinical trials, which are still lacking in literature.

  15. Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment.

    Science.gov (United States)

    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.

  16. Effects of extracellular fiber architecture on cell membrane shear stress in a 3D fibrous matrix.

    Science.gov (United States)

    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.

  17. Carboxy-terminal modulator protein attenuated extracellular matrix deposit by inhibiting phospho-Akt, TGF-β1 and α-SMA in kidneys of diabetic mice.

    Science.gov (United States)

    Chen, Ning; Hao, Jun; Li, Lisha; Li, Fan; Liu, Shuxia; Duan, Huijun

    2016-06-10

    Glomerulosclerosis and tubular interstitial extracellular matrix deposit and fibrosis are the main features of diabetic nephropathy, which are mediated by activation of PI3K/Akt signal pathway. Carboxy-terminal modulator protein (CTMP) is known as a negative regulator of PI3K/Akt pathway. Whether CTMP regulates renal extracellular matrix metabolism of diabetic nephropathy is still not known. Here, renal decreased CTMP, enhanced phospho-Akt (Ser 473), TGF-β1, α-SMA and extracellular matrix deposit are found in diabetic mice. Furthermore, high glucose decreases CTMP expression accompanied by enhanced phospho-Akt (Ser 473), TGF-β1 and α-SMA in cultured human renal proximal tubular epithelial cells (HKC), which are effectively prevented by transfection of pYr-ads-4-musCTMP vector. Moreover, delivery of pYr-ads-4-musCTMP vector into kidneys via tail vein of diabetic mice increases CTMP expression by 8.84 times followed by 60.00%, 76.50% and 24.37% decreases of phospho-Akt (Ser 473), TGF-β1 and α-SMA compared with diabetic mice receiving pYr-adshuttle-4 vector. Again, increased renal extracellular matrix accumulation of diabetic mice is also inhibited with delivery of pYr-ads-4-musCTMP vector. Our results indicate that CTMP attenuates renal extracellular matrix deposit by regulating the phosphorylation of Akt, TGF-β1 and α-SMA expression in diabetic mice. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. High resolution three-dimensional reconstruction of fibrotic skeletal muscle extracellular matrix.

    Science.gov (United States)

    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

  19. Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

    Directory of Open Access Journals (Sweden)

    Gustavo A. Nari

    2013-03-01

    Full Text Available Introduction: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. Objective: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. Methods: a combination of physical, chemical (Triton X-100 and SDS and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy and biochemical (DNA quantification studies. Results: the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin-eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde's staining demonstrated the conservation of reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson's capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. Conclusions: the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding.

  20. An investigation of the influence of extracellular matrix anisotropy and cell–matrix interactions on tissue architecture

    KAUST Repository

    Dyson, R. J.

    2015-09-02

    © 2015 Springer-Verlag Berlin Heidelberg Mechanical interactions between cells and the fibrous extracellular matrix (ECM) in which they reside play a key role in tissue development. Mechanical cues from the environment (such as stress, strain and fibre orientation) regulate a range of cell behaviours, including proliferation, differentiation and motility. In turn, the ECM structure is affected by cells exerting forces on the matrix which result in deformation and fibre realignment. In this paper we develop a mathematical model to investigate this mechanical feedback between cells and the ECM. We consider a three-phase mixture of collagen, culture medium and cells, and formulate a system of partial differential equations which represents conservation of mass and momentum for each phase. This modelling framework takes into account the anisotropic mechanical properties of the collagen gel arising from its fibrous microstructure. We also propose a cell–collagen interaction force which depends upon fibre orientation and collagen density. We use a combination of numerical and analytical techniques to study the influence of cell–ECM interactions on pattern formation in tissues. Our results illustrate the wide range of structures which may be formed, and how those that emerge depend upon the importance of cell–ECM interactions.

  1. The Notch pathway mediates the angiotensin II-induced synthesis of extracellular matrix components in podocytes.

    Science.gov (United States)

    Yao, Min; Wang, Xiaomei; Wang, Xiaomeng; Zhang, Tao; Chi, Yanqing; Gao, Feng

    2015-07-01

    The Notch pathway is known to contribute to the development of glomerular disease. Angiotensin II (Ang II), an important member of the renin-angiotensin system, stimulates the accumulation of extracellular matrix components in glomerular disease; however, the exact mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of the Notch pathway on the synthesis of extracellular matrix components in Ang II-stimulated podocytes. Mouse podocytes were stimulated with Ang II (10-6 mol/l). The activation of the Notch pathway was inhibited by a vector carrying short hairpin RNA (shRNA) targeting Notch1 (sh-Notch1) or by γ-secretase inhibitor (GSI). The protein levels of Notch1, Notch intracellular domain 1 (NICD1), hairy and enhancer of split-1 (Hes1), matrix metalloproteinase (MMP)-2, MMP-9, transforming growth factor-β1 (TGF-β1), type IV collagen and laminin were determined by western blot analysis. The Notch1, Hes1, MMP-2, MMP-9, TGF-β1, type IV collagen and laminin mRNA levels were detected by RT-PCR. The MMP-2 and MMP-9 activity was measured using a cell active fluorescence assay kit. The levels of TGF-β1, type IV collagen and laminin were determined in the culture medium of the podocytes by enzyme-linked immunosorbent assay (ELISA). Our results revealed that Ang II upregulated Notch1, NICD1, Hes1, TGF-β1, type IV collagen and laminin expression and downregulated MMP-2 and MMP-9 expression in the cultured podocytes. The inhibition of the Notch pathway by sh-Notch1 or GSI increased MMP-2 and MMP-9 expression, decreased the TGF-β1 level and suppressed type IV collagen and laminin expression. The inhibition of the Notch pathway by sh-Notch1 or GSI also increased MMP-2 and MMP-9 activity, and decreased TGF-β1 levels, type IV collagen levels and laminin secretion. These findings indicate that the Notch pathway potentially mediates the Ang II-induced synthesis of extracellular matrix components in podocytes through the

  2. Migration of tumor cells and leukocytes in extracellular matrix : proteolytic and nonproteolytic strategies for overcoming tissue barriers

    OpenAIRE

    Wolf, Katarina

    2003-01-01

    The extracellular matrix within connective tissues represents a structural scaffold as well as a barrier for motile cells, such as invading tumor cells or passenger leukocytes. It remains unclear how different cell types utilize matrix-degrading enzymes for proteolytic migration strategies and, on the other hand, non-proteolytic strategies to overcome 3D fibrillar matrix networks. To monitor cell migration, a 3D collagen model in vitro or the mouse dermis in vivo were used, in combination wit...

  3. Design and characterization of artificial extracellular matrix proteins for use as small-diameter vascular grafts

    Science.gov (United States)

    Heilshorn, Sarah

    Synthetic small-diameter vascular grafts often fail within three years of implantation. The underlying causes of graft failure are thought to be (i) a mismatch in the mechanical properties between the graft and host material and (ii) an inability of the graft to support the adhesion of endothelial cells. To address these two issues, artificial extracellular matrix (aECM) proteins contain elastin-like regions to provide physical integrity and cell-binding domains derived from fibronectin to promote endothelial cell attachment. Using recombinant protein technology, a family of artificial proteins was created with differing ratios of elastin-like regions to cell-binding domains, with variable placement of amino acid crosslinking residues, and with differing identity of cell-binding domain. Human umbilical vein endothelial cells (HUVEC) adhere in a sequence-specific manner to aECM proteins, secrete basal levels of key fibrinolytic regulators, and are capable of resisting a physiologically relevant detachment force. HUVEC spread more quickly and adhere more firmly to aECM proteins that contain the RGD versus the CS5 cell-binding domain. Decreasing the density of RGD cell-binding domains results in decreased HUVEC adhesion. Furthermore, amino acid selection even at sites up to 16 residues away from the cell-binding domain impacts HUVEC spreading and adhesion. HUVEC also adhere more strongly to stiffer aECM films. Therefore, the identity, density, and context of the cell-binding domain as well as the elastic modulus of the substrate are all important variables in influencing cell-substrate interactions. Proper amino acid sequence choice also influences the susceptibility of aECM proteins to elastase proteolysis; modifying 3% of the amino acid side chains results in a 7-fold reduction in degradation rate. An alternative strategy to decrease degradation involves incorporation of the noncanonical amino acid, 5,5,5-trifluoroisoleucine, into the favored proteolytic cut site

  4. The effect on the extracellular matrix of the deep fascia in response to leg lengthening

    Science.gov (United States)

    Wang, Hai-Qiang; Li, Xin-Kui; Wu, Zi-Xiang; Wei, Yi-Yong; Luo, Zhuo-Jing

    2008-01-01

    Background Whereas the alterations of diverse tissues in cellular and molecular levels have been investigated during leg lengthening via microscopy and biochemical studies, little is known about the response of deep fascia. This study aims to investigate the changes of the extracellular matrix in deep fascia in response to leg lengthening. Methods Animal model of leg lengthening was established in New Zealand white rabbits. Distraction was initiated at a rate of 1 mm/day and 2 mm/day in two steps, and preceded until increases of 10% and 20% in the initial length of tibia had been achieved. Alcian blue stain and picrosirius-polarization method were used for the study of the extracellular matrix of deep fascia samples. Leica DM LA image analysis system was used to investigate the quantitative changes of collagen type I and III. Results Alcian blue stain showed that glycosaminoglycans of fascia of each group were composed of chondroitin sulphate and heparin sulphate, but not of keratan sulphate. Under the polarization microscopy, the fascia consisted mainly of collagen type I. After leg lengthening, the percentage of collagen type III increased. The most similar collagen composition of the fascia to that of the normal fascia was detected at a 20% increase in tibia length achieved via a distraction rate of 1 mm/d. Conclusion The changes in collagen distribution and composition occur in deep fascia during leg lengthening. Although different lengthening schemes resulted in varied matrix changes, the most comparable collagen composition to be demonstrated under the scheme of a distraction rate of 1 mm/day and 20% increase in tibia length. Efficient fascia regeneration is initiated only in certain combinations of the leg load parameters including appropriate intensity and duration time, e.g., either low density distraction that persist a relatively short time or high distraction rates. PMID:18611283

  5. PPARdelta promotes wound healing by up-regulating TGF-beta1-dependent or -independent expression of extracellular matrix proteins.

    Science.gov (United States)

    Ham, Sun Ah; Kim, Hyo Jung; Kim, Hyun Joon; Kang, Eun Sil; Eun, So Young; Kim, Gil Hyeong; Park, Myung Hyun; Woo, Im Sun; Kim, Hye Jung; Chang, Ki Churl; Lee, Jae Heun; Seo, Han Geuk

    2010-06-01

    Although the peroxisome proliferator-activated receptor (PPAR) delta has been implicated in the wound healing process, its exact role and mechanism of action have not been fully elucidated. Our previous findings showed that PPARdelta induces the expression of the transforming growth factor (TGF)-beta1, which has been implicated in the deposit of extracellular matrix proteins. Here, we demonstrate that administration of GW501516, a specific PPARdelta ligand, significantly promoted wound closure in the experimental mouse and had a profound effect on the expression of collagen types I and III, alpha-smooth muscle actin, pSmad3 and TGF-beta1, which play a pivotal role in wound healing processes. Activation of PPARdelta increased migration of human epidermal keratinocytes and dermal fibroblasts in in vitro scrape-wounding assays. Addition of a specific ALK5 receptor inhibitor SB431542 significantly suppressed GW501516-induced migration of human keratinocytes and fibroblasts. In these cells, activated PPARdelta also induced the expression of collagen types I and III and fibronectin in a TGF-beta1-dependent or -independent manner. The effect of PPARdelta on the expression of type III collagen was dually regulated by the direct binding of PPARdelta and Smad3 to a direct repeat-1 site and a Smad-binding element, respectively, of the type III gene promoter. Taken together, these results demonstrated that PPARdelta plays an important role in skin wound healing in vivo and that it functions by accelerating extracellular matrix-mediated cellular interactions in a process mediated by the TGF-beta1/Smad3 signaling-dependent or - independent pathway.

  6. Elastic extracellular matrix of the embryonic chick heart: an immunohistological study using laser confocal microscopy.

    Science.gov (United States)

    Hurle, J M; Kitten, G T; Sakai, L Y; Volpin, D; Solursh, M

    1994-08-01

    The "elastic matrix" constitutes a specialized component of the extracellular matrix which confers resiliency to tissues and organs subjected to repeated deformations. The role of the elastic matrix in living organisms appears to be of key importance since diseases characterized by expression of defective inherited genes which encode components of the elastic matrix lead to premature death. While the elastic matrix of adult organs has received a great deal of attention, little is known about when it first appears in embryonic tissues or its possible role in developing organs. In the present study we have performed an immunohistochemical study of the distribution of elastin and three additional components often associated with elastic matrices in adult tissues (i.e., fibrillin, emilin, and type VI collagen) during the development of the chicken embryonic heart. The three-dimensional arrangement of these components was established through the observation of whole-amount specimens with scanning laser confocal microscopy. Our results revealed three different periods of heart development regarding the composition of the elastic matrix. Prior to stage 21 the embryonic heart lacks elastin but exhibits a matrix scaffold of fibrillin and emilin associated with the endocardium and the developing cardiac jelly. Between stages 22 and 29 the heart shows a transient elastic scaffold in the outflow tract which contains elastin, fibrillin, and emilin. Elastin-positive fibrillar material is also observed during these stages in the base of the atrioventricular cushion adjacent to the myocardial wall. In addition, emilin-positive material appears to be associated with the zones of formation of ventricular trabeculae. Collagen type VI was not detected during these early stages. From stage 30 to stage 40 a progressive modification of the pattern of distribution of elastin, fibrillin, emilin, and collagen type VI is observed in association with the formation of the definitive four

  7. The potential for targeting extracellular LOX proteins in human malignancy

    Directory of Open Access Journals (Sweden)

    Mayorca-Guiliani A

    2013-11-01

    Full Text Available Alejandro Mayorca-Guiliani, Janine T Erler Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Abstract: The extracellular matrix (ECM is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX enzymes cross-links collagens and elastin and, therefore, is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient deaths. However, LOX biosynthesis results in by-product with antiproliferative properties in certain cancers, and LOX enzymes may have different effects depending on the molecular network in which they are active. Therefore, the design of therapies targeting the LOX family needs to be guided by the molecular makeup of the individual disease and will probably require other agents to act on both the LOX enzymes and their associated network. Keywords: cancer, extracellular matrix, lysyl oxidase, metastasis

  8. Expression of Extracellular Matrix Proteins in Basal Membranes During Fetal Nephron Development in Mice

    Directory of Open Access Journals (Sweden)

    Beyhan GÜRCÜ

    2016-04-01

    Full Text Available In this study, we investigated the distribution of laminin, collagen type IV, nidogen and fibronectine during metanephric development in fetal mouse kidney by immunohistochemistry. Stain density of basement membranes of tubules, glomerules and mesangial matrix were compared in pre-capillary, immature glomerular and mature glomerular stages of fetal kidney. All the matrix proteins were strongly stained in precapillary stage. In immature glomerular stage, a strong staining was observed for fibronectin. Staining intensity was slightly decreased for the other proteins in this stage. In mature glomerular stage, diminished staining for all proteins was observed similar to the previous stage, except fibronectin. The strongest immunoreactions were found for fibronectin and nidogen in all investigated stages. In general, there was a similar staining intensity for all glycoproteins during maturation except for laminin. It was thought that the distribution of extracellular matrix molecules plays an important role for the kidney development. Interactions amoung these molecules probably crucial on cell behavior like migration, proliferation and differentiation in normal development of the nephron.

  9. Epilysin (MMP-28) is deposited to the basolateral extracellular matrix of epithelial cells.

    Science.gov (United States)

    Heiskanen, Tuomas J; Illman, Sara A; Lohi, Jouko; Keski-Oja, Jorma

    2009-03-01

    Epilysin (MMP-28) is a conserved member of the matrix metalloproteinase (MMP) family. It is expressed in various normal tissues, and induced in wounds and in developing and regenerating nerves. Epilysin induces TGF-beta mediated epithelial to mesenchymal transition, but its other functions are largely unknown. We have characterized the localization of both catalytically active and mutated inactive, overexpressed epilysin in established epithelial cell lines. We found that epilysin was localized abundantly to the basolateral side of the cells and associated with the extracellular matrix (ECM) as verified by immunoblotting and confocal microscopy. Overexpression of epilysin in MDCK cells resulted in a drastic reduction of basolateral ECM, as observed by the disappearance of collagen type IV, laminin and fibronectin. Cultivation of epilysin expressing MDCK cells in defined serum free medium resulted in the restoration of these proteins to the ECM. The levels of fibronectin and collagen IV were, however, reduced in epilysin expressing cells under the serum free conditions, and degradation fragments of collagen IV were detected supporting the activation of proteolysis by epilysin. Epilysin was observed in its unprocessed 50 kDa active form in the ECM of MDCK cells under serum free conditions whereas in cells cultured in serum containing it was processed to the 48 kDa form. Current results indicate that epilysin associates with the basolateral ECM of cultured epithelial cells, where it plausibly plays a role in the regulation of matrix composition and turnover.

  10. Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors

    Directory of Open Access Journals (Sweden)

    Daniel Santos

    2016-12-01

    Full Text Available After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM components and neurotrophic factors (NTFs exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF/neurotrophin-3 (NT3 or collagen, fibronectin and brain-derived neurotrophic factor (BDNF selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid (PLGA microspheres (MP (LM + MP.NGF/NT3, or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF. Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation.

  11. Interdigital cell death in the embryonic limb is associated with depletion of Reelin in the extracellular matrix

    OpenAIRE

    2013-01-01

    Interdigital cell death is a physiological regression process responsible for sculpturing the digits in the embryonic vertebrate limb. Changes in the intensity of this degenerative process account for the different patterns of interdigital webbing among vertebrate species. Here, we show that Reelin is present in the extracellular matrix of the interdigital mesoderm of chick and mouse embryos during the developmental stages of digit formation. Reelin is a large extracellular glycoprotein which...

  12. Static stretch affects neural stem cell differentiation in an extracellular matrix-dependent manner

    Science.gov (United States)

    Arulmoli, Janahan; Pathak, Medha M.; McDonnell, Lisa P.; Nourse, Jamison L.; Tombola, Francesco; Earthman, James C.; Flanagan, Lisa A.

    2015-02-01

    Neural stem and progenitor cell (NSPC) fate is strongly influenced by mechanotransduction as modulation of substrate stiffness affects lineage choice. Other types of mechanical stimuli, such as stretch (tensile strain), occur during CNS development and trauma, but their consequences for NSPC differentiation have not been reported. We delivered a 10% static equibiaxial stretch to NSPCs and examined effects on differentiation. We found static stretch specifically impacts NSPC differentiation into oligodendrocytes, but not neurons or astrocytes, and this effect is dependent on particular extracellular matrix (ECM)-integrin linkages. Generation of oligodendrocytes from NSPCs was reduced on laminin, an outcome likely mediated by the α6 laminin-binding integrin, whereas similar effects were not observed for NSPCs on fibronectin. Our data demonstrate a direct role for tensile strain in dictating the lineage choice of NSPCs and indicate the dependence of this phenomenon on specific substrate materials, which should be taken into account for the design of biomaterials for NSPC transplantation.

  13. Cells must express components of the planar cell polarity system and extracellular matrix to support cytonemes.

    Science.gov (United States)

    Huang, Hai; Kornberg, Thomas B

    2016-09-03

    Drosophila dorsal air sac development depends on Decapentaplegic (Dpp) and Fibroblast growth factor (FGF) proteins produced by the wing imaginal disc and transported by cytonemes to the air sac primordium (ASP). Dpp and FGF signaling in the ASP was dependent on components of the planar cell polarity (PCP) system in the disc, and neither Dpp- nor FGF-receiving cytonemes extended over mutant disc cells that lacked them. ASP cytonemes normally navigate through extracellular matrix (ECM) composed of collagen, laminin, Dally and Dally-like (Dlp) proteins that are stratified in layers over the disc cells. However, ECM over PCP mutant cells had reduced levels of laminin, Dally and Dlp, and whereas Dpp-receiving ASP cytonemes navigated in the Dally layer and required Dally (but not Dlp), FGF-receiving ASP cytonemes navigated in the Dlp layer, requiring Dlp (but not Dally). These findings suggest that cytonemes interact directly and specifically with proteins in the stratified ECM.

  14. Extracellular matrix proteins as temporary coating for thin-film neural implants

    Science.gov (United States)

    Ceyssens, Frederik; Deprez, Marjolijn; Turner, Neill; Kil, Dries; van Kuyck, Kris; Welkenhuysen, Marleen; Nuttin, Bart; Badylak, Stephen; Puers, Robert

    2017-02-01

    Objective. This study investigates the suitability of a thin sheet of extracellular matrix (ECM) proteins as a resorbable coating for temporarily reinforcing fragile or ultra-low stiffness thin-film neural implants to be placed on the brain, i.e. microelectrocorticographic (µECOG) implants. Approach. Thin-film polyimide-based electrode arrays were fabricated using lithographic methods. ECM was harvested from porcine tissue by a decellularization method and coated around the arrays. Mechanical tests and an in vivo experiment on rats were conducted, followed by a histological tissue study combined with a statistical equivalence test (confidence interval approach, 0.05 significance level) to compare the test group with an uncoated control group. Main results. After 3 months, no significant damage was found based on GFAP and NeuN staining of the relevant brain areas. Significance. The study shows that ECM sheets are a suitable temporary coating for thin µECOG neural implants.

  15. Extracellular matrix macromolecules: potential tools and targets in cancer gene therapy.

    Science.gov (United States)

    Sainio, Annele; Järveläinen, Hannu

    2014-01-01

    Tumour cells create their own microenvironment where they closely interact with a variety of soluble and non-soluble molecules, different cells and numerous other components within the extracellular matrix (ECM). Interaction between tumour cells and the ECM is bidirectional leading to either progression or inhibition of tumourigenesis. Therefore, development of novel therapies targeted primarily to tumour microenvironment (TME) is highly rational. Here, we give a short overview of different macromolecules of the ECM and introduce mechanisms whereby they contribute to tumourigenesis within the TME. Furthermore, we present examples of individual ECM macromolecules as regulators of cell behaviour during tumourigenesis. Finally, we focus on novel strategies of using ECM macromolecules as tools or targets in cancer gene therapy in the future.

  16. Haemodynamic and extracellular matrix cues regulate the mechanical phenotype and stiffness of aortic endothelial cells.

    Science.gov (United States)

    Collins, Caitlin; Osborne, Lukas D; Guilluy, Christophe; Chen, Zhongming; O'Brien, E Tim; Reader, John S; Burridge, Keith; Superfine, Richard; Tzima, Ellie

    2014-06-11

    Endothelial cells (ECs) lining blood vessels express many mechanosensors, including platelet endothelial cell adhesion molecule-1 (PECAM-1), that convert mechanical force into biochemical signals. While it is accepted that mechanical stresses and the mechanical properties of ECs regulate vessel health, the relationship between force and biological response remains elusive. Here we show that ECs integrate mechanical forces and extracellular matrix (ECM) cues to modulate their own mechanical properties. We demonstrate that the ECM influences EC response to tension on PECAM-1. ECs adherent on collagen display divergent stiffening and focal adhesion growth compared with ECs on fibronectin. This is because of protein kinase A (PKA)-dependent serine phosphorylation and inactivation of RhoA. PKA signalling regulates focal adhesion dynamics and EC compliance in response to shear stress in vitro and in vivo. Our study identifies an ECM-specific, mechanosensitive signalling pathway that regulates EC compliance and may serve as an atheroprotective mechanism that maintains blood vessel integrity in vivo.

  17. Extracellular matrix-inspired growth factor delivery systems for bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Martino, Mikaël M. [Osaka Univ. (Japan). Immunology Frontier Research Center; Briquez, Priscilla S. [Ecole Polytechnique Federale de Lausanne (Switzerland). Inst. of Bioengineering; Maruyama, Kenta [Osaka Univ. (Japan). Immunology Frontier Research Center; Hubbell, Jeffrey A. [Ecole Polytechnique Federale de Lausanne (Switzerland). Inst. of Bioengineering; Univ. of Chicago, IL (United States). Inst. for Molecular Engineering; Argonne National Lab. (ANL), Argonne, IL (United States)

    2015-04-17

    Growth factors are very promising molecules to enhance bone regeneration. However, their translation to clinical use has been seriously limited, facing issues related to safety and cost-effectiveness. These problems derive from the vastly supra-physiological doses of growth factor used without optimized delivery systems. Therefore, these issues have motivated the development of new delivery systems allowing better control of the spatio-temporal release and signaling of growth factors. Because the extracellular matrix (ECM) naturally plays a fundamental role in coordinating growth factor activity in vivo, a number of novel delivery systems have been inspired by the growth factor regulatory function of the ECM. After introducing the role of growth factors during the bone regeneration process, this review exposes different issues that growth factor-based therapies have encountered in the clinic and highlights recent delivery approaches based on the natural interaction between growth factor and the ECM.

  18. A non-equilibrium thermodynamic model for tumor extracellular matrix with enzymatic degradation

    Science.gov (United States)

    Xue, Shi-Lei; Li, Bo; Feng, Xi-Qiao; Gao, Huajian

    2017-07-01

    The extracellular matrix (ECM) of a solid tumor not only affords scaffolding to support tumor architecture and integrity but also plays an essential role in tumor growth, invasion, metastasis, and therapeutics. In this paper, a non-equilibrium thermodynamic theory is established to study the chemo-mechanical behaviors of tumor ECM, which is modeled as a poroelastic polyelectrolyte consisting of a collagen network and proteoglycans. By using the principle of maximum energy dissipation rate, we deduce a set of governing equations for drug transport and mechanosensitive enzymatic degradation in ECM. The results reveal that osmosis is primarily responsible for the compression resistance of ECM. It is suggested that a well-designed ECM degradation can effectively modify the tumor microenvironment for improved efficiency of cancer therapy. The theoretical predictions show a good agreement with relevant experimental observations. This study aimed to deepen our understanding of tumor ECM may be conducive to novel anticancer strategies.

  19. Extracellular matrix considerations for scar-free repair and regeneration: insights from regenerative diversity among vertebrates.

    Science.gov (United States)

    Godwin, James; Kuraitis, Drew; Rosenthal, Nadia

    2014-11-01

    The extracellular matrix (ECM) is an essential feature of development, tissue homeostasis and recovery from injury. How the ECM responds dynamically to cellular and soluble components to support the faithful repair of damaged tissues in some animals but leads to the formation of acellular fibrotic scar tissue in others has important clinical implications. Studies in highly regenerative organisms such as the zebrafish and the salamander have revealed a specialist formulation of ECM components that support repair and regeneration, while avoiding scar tissue formation. By comparing a range of different contexts that feature scar-less healing and full regeneration vs. scarring through fibrotic repair, regenerative therapies that incorporate ECM components could be significantly enhanced to improve both regenerative potential and functional outcomes. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.

  20. Biomaterials approaches to modeling macrophage-extracellular matrix interactions in the tumor microenvironment.

    Science.gov (United States)

    Springer, Nora L; Fischbach, Claudia

    2016-08-01

    Tumors are characterized by aberrant extracellular matrix (ECM) remodeling and chronic inflammation. While advances in biomaterials and tissue engineering strategies have led to important new insights regarding the role of ECM composition, structure, and mechanical properties in cancer in general, the functional link between these parameters and macrophage phenotype is poorly understood. Nevertheless, increasing experimental evidence suggests that macrophage behavior is similarly controlled by physicochemical properties of the ECM and consequential changes in mechanosignaling. Here, we will summarize the current knowledge of macrophage biology and ECM-mediated differences in mechanotransduction and discuss future opportunities of biomaterials and tissue engineering platforms to interrogate the functional relationship between these parameters and their relevance to cancer.

  1. Engineering the extracellular matrix for clinical applications: endoderm, mesoderm, and ectoderm.

    Science.gov (United States)

    Williams, Miguel L; Bhatia, Sujata K

    2014-03-01

    Tissue engineering is rapidly progressing from a research-based discipline to clinical applications. Emerging technologies could be utilized to develop therapeutics for a wide range of diseases, but many are contingent on a cell scaffold that can produce proper tissue ultrastructure. The extracellular matrix, which a cell scaffold simulates, is not merely a foundation for tissue growth but a dynamic participant in cellular crosstalk and organ homeostasis. Cells change their growth rates, recruitment, and differentiation in response to the composition, modulus, and patterning of the substrate on which they reside. Cell scaffolds can regulate these factors through precision design, functionalization, and application. The ideal therapy would utilize highly specialized cell scaffolds to best mimic the tissue of interest. This paper discusses advantages and challenges of optimized cell scaffold design in the endoderm, mesoderm, and ectoderm for clinical applications in tracheal transplant, cardiac regeneration, and skin grafts, respectively. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    Science.gov (United States)

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

  3. Integrated extracellular matrix signaling in mammary gland development and breast cancer progression.

    Science.gov (United States)

    Zhu, Jieqing; Xiong, Gaofeng; Trinkle, Christine; Xu, Ren

    2014-09-01

    Extracellular matrix (ECM), a major component of the cellular microenvironment, plays critical roles in normal tissue morphogenesis and disease progression. Binding of ECM to membrane receptor proteins, such as integrin, discoidin domain receptors, and dystroglycan, elicits biochemical and biomechanical signals that control cellular architecture and gene expression. These ECM signals cooperate with growth factors and hormones to regulate cell migration, differentiation, and transformation. ECM signaling is tightly regulated during normal mammary gland development. Deposition and alignment of fibrillar collagens direct migration and invasion of mammary epithelial cells during branching morphogenesis. Basement membrane proteins are required for polarized acinar morphogenesis and milk protein expression. Deregulation of ECM proteins in the long run is sufficient to promote breast cancer development and progression. Recent studies demonstrate that the integrated biophysical and biochemical signals from ECM and soluble factors are crucial for normal mammary gland development as well as breast cancer progression.

  4. Biodegradation-Resistant Multilayers Coated with Gold Nanoparticles. Toward a Tailor-made Artificial Extracellular Matrix.

    Science.gov (United States)

    Prokopović, Vladimir Z; Vikulina, Anna S; Sustr, David; Duschl, Claus; Volodkin, Dmitry

    2016-09-21

    Polymer multicomponent coatings such as multilayers mimic an extracellular matrix (ECM) that attracts significant attention for the use of the multilayers as functional supports for advanced cell culture and tissue engineering. Herein, biodegradation and molecular transport in hyaluronan/polylysine multilayers coated with gold nanoparticles were described. Nanoparticle coating acts as a semipermeable barrier that governs molecular transport into/from the multilayers and makes them biodegradation-resistant. Model protein lysozyme (mimics of ECM-soluble signals) diffuses into the multilayers as fast- and slow-diffusing populations existing in an equilibrium. Such a composite system may have high potential to be exploited as degradation-resistant drug-delivery platforms suitable for cell-based applications.

  5. The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel.

    Science.gov (United States)

    Ahmed, Ahmed Ashour; Mills, Anthony D; Ibrahim, Ashraf E K; Temple, Jillian; Blenkiron, Cherie; Vias, Maria; Massie, Charlie E; Iyer, N Gopalakrishna; McGeoch, Adam; Crawford, Robin; Nicke, Barbara; Downward, Julian; Swanton, Charles; Bell, Stephen D; Earl, Helena M; Laskey, Ronald A; Caldas, Carlos; Brenton, James D

    2007-12-01

    The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

  6. Aristoyunnolin H attenuates extracellular matrix secretion in cardiac fibroblasts by inhibiting calcium influx.

    Science.gov (United States)

    Chen, Shao-Rui; Zhang, Wen-Ping; Bao, Jing-Mei; Cheng, Zhong-Bin; Yin, Sheng

    2017-01-01

    Aristoyunnolin H is a novel aristophyllene sesquiterpenoid isolated from the traditional Chinese medicine Aristolochia yunnanensis Franch. The present research was designed to explore the anti-fibrotic effects of aristoyunnolin H in adult rat cardiac fibroblasts (CFs) stimulated with angiotensin II (Ang II). Western blot analysis data showed that aristoyunnolin H reduced the upregulation of fibronectin (FN), connective tissue growth factor and collagen I(Col I) production induced by Ang II in CFs. By studying the dynamic intracellular changes of Ca(2+), we further found that while aristoyunnolin H relieved the calcium influx, it has no effect on intracellular calcium store release. Meanwhile, aristoyunnolin H also inhibited the Ang II-stimulated phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II. In conclusion, aristoyunnolin H may attenuate extracellular matrix secretion in vitro by inhibiting Ang II-induced calcium signaling.

  7. Effects of various extracellular matrix proteins on the growth of HL-1 cardiomyocytes.

    Science.gov (United States)

    Choi, Seongkyun; Hong, Yoonmi; Lee, Insu; Huh, Dongeun; Jeon, Tae-Joon; Kim, Sun Min

    2013-01-01

    We present the physical and biochemical effects of extracellular matrixes (ECMs) on HL-1 cardiomyocytes. ECMs play major roles in cell growth, adhesion and the maintenance of native cell functions. We investigated the effects of 6 different cell culture systems: 5 different ECM-treated surfaces (fibronectin, laminin, collagen I, gelatin and a gelatin/fibronectin mixture) and 1 nontreated surface. Surface morphology was scanned and analyzed using atomic force microscopy in order to investigate the physical effects of ECMs. The attachment, growth, viability, proliferation and phenotype of the cells were analyzed using phase-contrast microscopy and immunocytochemistry to elucidate the biochemical effects of ECMs. Our study provides basic information for understanding cell-ECM interactions and should be utilized in future cardiac cell research and tissue engineering.

  8. The Role of Structural Extracellular Matrix Proteins in Urothelial Bladder Cancer

    Directory of Open Access Journals (Sweden)

    Andrea Brunner

    2007-01-01

    Full Text Available The extracellular matrix (ECM plays a key role in the modulation of cancer cell invasion. In urothelial carcinoma of the bladder (UC the role of ECM proteins has been widely studied. The mechanisms, which are involved in the development of invasion, progression and generalization, are complex, depending on the interaction of ECM proteins with each other as well as with cancer cells. The following review will focus on the pathogenetic role and prognostic value of structural proteins, such as laminins, collagens, fi bronectin (FN, tenascin (Tn-C and thrombospondin 1 (TSP1 in UC. In addition, the role of integrins mediating the interaction of ECM molecules and cancer cells will be addressed, since integrin-mediated FN, Tn-C and TSP1 interactions seem to play an important role during tumor cell invasion and angiogenesis.

  9. Extra-cellular matrix proteins induce matrix metalloproteinase-1 (MMP-1 activity and increase airway smooth muscle contraction in asthma.

    Directory of Open Access Journals (Sweden)

    Natasha K Rogers

    Full Text Available Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM deposition. Matrix metalloproteinase-1 (MMP-1 is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the β1 and β3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms.

  10. Dentin extracellular matrix (ECM) proteins: comparison to bone ECM and contribution to dynamics of dentinogenesis.

    Science.gov (United States)

    Butler, William T; Brunn, Jan C; Qin, Chunlin

    2003-01-01

    Dentinogenesis involves the initial odontoblastic synthesis of a collagen-rich extracellular matrix (ECM) and predentin that is converted to dentin when the collagen fibrils become mineralized. Since the width of predentin is rather uniform, we postulate that extracellular events regulate dentinogenesis. Similarly, osteogenesis involves an initial unmineralized osteoid that is mineralized and converted to bone. To gain insights into these two processes, we compared ECM proteins in bone with those in dentin, focusing upon the sialic acid (SA)-rich proteins. We observed qualitative similarities between the SA-rich proteins, but distinct differences in the amounts of osteopontin (OPN) and dentin sialoprotein (DSP). OPN, a predominant protein in bone, was found in much smaller amounts in dentin. Conversely, DSP was abundant in dentin ECM, but found sparingly in bone. Molecular cloning experiments indicate that coding sequences for DSP and dentin phosphoprotein (DPP) are found on the same mRNA. We believe that the initial form of the precursor protein DSPP is inactive in influencing the mineralization process and that it must be activated by cleavage of peptide bonds in conserved regions. Thus, unknown proteinases would act on DSPP, possibly at the mineralization front, and liberate active DPP, which plays an initiation and regulatory role in the formation of apatite crystals. This post-translational processing reaction would represent an important control point in dentinogenesis. Recently, we identified uncleaved DSPP in dentin extracts, which should allow us to test portions of our hypothesis.

  11. A potential role for glia-derived extracellular matrix remodeling in postinjury epilepsy.

    Science.gov (United States)

    Kim, Soo Young; Porter, Brenda E; Friedman, Alon; Kaufer, Daniela

    2016-09-01

    Head trauma and vascular injuries are known risk factors for acquired epilepsy. The sequence of events that lead from the initial injury to the development of epilepsy involves complex plastic changes and circuit rewiring. In-depth, comprehensive understanding of the epileptogenic process is critical for the identification of disease-modifying targets. Here we review the complex interactions of cellular and extracellular components that may promote epileptogenesis, with an emphasis on the role of astrocytes. Emerging evidence demonstrates that astrocytes promptly respond to brain damage and play a critical role in the development of postinjury epilepsy. Astrocytes have been shown to regulate extracellular matrix (ECM) remodeling, which can affect plasticity and stability of synapses and, in turn, contribute to the epileptogenic process. From these separate lines of evidence, we present a hypothesis suggesting a possible role for astrocyte-regulated remodeling of ECM and perineuronal nets, a specialized ECM structure around fast-spiking inhibitory interneurons, in the development and progression of posttraumatic epilepsies. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Novel thermostable glycosidases in the extracellular matrix of the terrestrial cyanobacterium Nostoc commune.

    Science.gov (United States)

    Morsy, Fatthy Mohamed; Kuzuha, Satomi; Takani, Yayoi; Sakamoto, Toshio

    2008-10-01

    The cyanobacterium Nostoc commune is adapted to the terrestrial environment and forms a visible colony in which the cells are embedded in extracellular polysaccharides (EPSs), which play a crucial role in the extreme desiccation tolerance of this organism. When natural colonies were immersed in water, degradation of the colonies occurred within 2 days and N. commune cells were released into the water. The activities that hydrolyze glycoside bonds in various N. commune fractions were examined using artificial nitrophenyl-linked sugars as substrates. A beta-D-glucosidase purified from the water-soluble fraction was resistant to 20 min of boiling. The beta-D-glucosidase, with a molecular mass of 20 kDa, was identified as a cyanobacterial fasciclin protein based on its N-terminal amino-acid sequence. The 36-kDa major protein in the water-soluble fraction was purified, and the N-terminal amino-acid sequence of the protein was found to be identical to that of the water-stress protein (WspA) of N. commune. This WspA protein also showed heat-resistant beta-D-galactosidase activity. The fasciclin protein and WspA in the extracellular matrix may play a role in the hydrolysis of the EPSs surrounding the cells, possibly as an aid in the dispersal of cells, thus expanding the colonies of this cyanobacterium.

  13. Differential Expression of Extracellular Matrix and Growth Factors by Embryoid Bodies in Hydrodynamic and Static Cultures

    Science.gov (United States)

    Fridley, Krista M.; Nair, Rekha

    2014-01-01

    During development, cell fate specification and tissue development are orchestrated by the sequential presentation of soluble growth factors (GF) and extracellular matrix (ECM) molecules. Similarly, differentiation of stem cells in vitro relies upon the temporal presence of extracellular cues within the microenvironment. Hydrodynamic culture systems are not limited by volume restrictions and therefore offer several practical advantages for scalability over static cultures; however, hydrodynamic cultures expose cells to physical parameters not present in static culture, such as fluid shear stress and mass transfer through convective forces. In this study, the differences between static and hydrodynamic culture conditions on the expression of ECM and GF molecules during the differentiation of mouse embryonic stem cells were examined at both the gene and protein level. The expression of ECM and GF genes exhibited an early decrease in static cultures based on heat map and hierarchical clustering analysis and a relative delayed increase in hydrodynamic cultures. Although the temporal patterns of specific ECM and GF protein expression were comparable between static and hydrodynamic cultures, several notable differences in the magnitudes of expression were observed at similar time points. These results describe the establishment of an analytical framework that can be used to examine the expression patterns of ECM and GF molecules expressed by pluripotent stem cells undergoing differentiation as 3D multicellular aggregates under different culture conditions, and suggest that physical parameters of stem cell microenvironments can alter endogenous ECM and GF expression profiles that may, in turn, influence cell fate decisions. PMID:25423310

  14. Myogenic Progenitor Cells Control Extracellular Matrix Production by Fibroblasts during Skeletal Muscle Hypertrophy.

    Science.gov (United States)

    Fry, Christopher S; Kirby, Tyler J; Kosmac, Kate; McCarthy, John J; Peterson, Charlotte A

    2017-01-05

    Satellite cells, the predominant stem cell population in adult skeletal muscle, are activated in response to hypertrophic stimuli and give rise to myogenic progenitor cells (MPCs) within the extracellular matrix (ECM) that surrounds myofibers. This ECM is composed largely of collagens secreted by interstitial fibrogenic cells, which influence satellite cell activity and muscle repair during hypertrophy and aging. Here we show that MPCs interact with interstitial fibrogenic cells to ensure proper ECM deposition and optimal muscle remodeling in response to hypertrophic stimuli. MPC-dependent ECM remodeling during the first week of a growth stimulus is sufficient to ensure long-term myofiber hypertrophy. MPCs secrete exosomes containing miR-206, which represses Rrbp1, a master regulator of collagen biosynthesis, in fibrogenic cells to prevent excessive ECM deposition. These findings provide insights into how skeletal stem and progenitor cells interact with other cell types to actively regulate their extracellular environments for tissue maintenance and adaptation. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Ischemic injury leads to extracellular matrix alterations in retina and optic nerve

    Science.gov (United States)

    Reinhard, Jacqueline; Renner, Marina; Wiemann, Susanne; Shakoor, Daniel A.; Stute, Gesa; Dick, H. Burkhard; Faissner, Andreas; Joachim, Stephanie C.

    2017-01-01

    Retinal ischemia occurs in a variety of eye diseases. Restrained blood flow induces retinal damage, which leads to progressive optic nerve degeneration and vision loss. Previous studies indicate that extracellular matrix (ECM) constituents play an important role in complex tissues, such as retina and optic nerve. They have great impact on de- and regeneration processes and represent major candidates of central nervous system glial scar formation. Nevertheless, the importance of the ECM during ischemic retina and optic nerve neurodegeneration is not fully understood yet. In this study, we analyzed remodeling of the extracellular glycoproteins fibronectin, laminin, tenascin-C and tenascin-R and the chondroitin sulfate proteoglycans (CSPGs) aggrecan, brevican and phosphacan/RPTPβ/ζ in retinae and optic nerves of an ischemia/reperfusion rat model via quantitative real-time PCR, immunohistochemistry and Western blot. A variety of ECM constituents were dysregulated in the retina and optic nerve after ischemia. Regarding fibronectin, significantly elevated mRNA and protein levels were observed in the retina following ischemia, while laminin and tenascin-C showed enhanced immunoreactivity in the optic nerve after ischemia. Interestingly, CSPGs displayed significantly increased expression levels in the optic nerve. Our study demonstrates a dynamic expression of ECM molecules following retinal ischemia, which strengthens their regulatory role during neurodegeneration. PMID:28262779

  16. Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

    2006-05-25

    Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

  17. Biological conduits combining bone marrow mesenchymal stem cells and extracellular matrix to treat long-segment sciatic nerve defects

    Directory of Open Access Journals (Sweden)

    Yang Wang

    2015-01-01

    Full Text Available The transplantation of polylactic glycolic acid conduits combining bone marrow mesenchymal stem cells and extracellular matrix gel for the repair of sciatic nerve injury is effective in some respects, but few data comparing the biomechanical factors related to the sciatic nerve are available. In the present study, rabbit models of 10-mm sciatic nerve defects were prepared. The rabbit models were repaired with autologous nerve, a polylactic glycolic acid conduit + bone marrow mesenchymal stem cells, or a polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel. After 24 weeks, mechanical testing was performed to determine the stress relaxation and creep parameters. Following sciatic nerve injury, the magnitudes of the stress decrease and strain increase at 7,200 seconds were largest in the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel group, followed by the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells group, and then the autologous nerve group. Hematoxylin-eosin staining demonstrated that compared with the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells group and the autologous nerve group, a more complete sciatic nerve regeneration was found, including good myelination, regularly arranged nerve fibers, and a completely degraded and resorbed conduit, in the polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel group. These results indicate that bridging 10-mm sciatic nerve defects with a polylactic glycolic acid conduit + bone marrow mesenchymal stem cells + extracellular matrix gel construct increases the stress relaxation under a constant strain, reducing anastomotic tension. Large elongations under a constant physiological load can limit the anastomotic opening and shift, which is beneficial for the regeneration and functional reconstruction of sciatic nerve. Better

  18. The effect of stromelysin-1 (MMP-3) on non-collagenous extracellular matrix proteins of demineralized dentin and the adhesive properties of restorative resins.

    Science.gov (United States)

    Boukpessi, T; Menashi, S; Camoin, L; Tencate, J M; Goldberg, M; Chaussain-Miller, C

    2008-11-01

    Dentin non-collagenous matrix components (NCPs) are structural proteins involved in the formation, the architecture and the mineralization of the extracellular matrix (ECM). We investigated here how recombinant metalloproteinase stromelysin-1, also termed MMP-3, initiates the release of ECM molecules from artificially demineralized human dentin. Analysis of the supernatants by Western blotting reveals that MMP-3 extracts PGs (decorin, biglycan), and also a series of phosphorylated proteins: dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP) and MEPE, but neither dentin matrix protein-1 (DMP1), another member of the SIBLING family, nor osteocalcin (OC), a non-phosphorylated matrix molecule. After treatment of dentin surfaces by MMP-3, scanning electron microscope (SEM) examination of resin replica shows an increased penetration of the resin into the dentin tubules when compared to surfaces only treated by demineralizing solutions. This preclinical investigation suggests that MMP-3 may be used to improve the adhesive properties of restorative materials.

  19. The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain

    Institute of Scientific and Technical Information of China (English)

    Tosikazu AMANO; Olivia KWAK; Liezhen FU; Anastasia MARSHAK; Yun-Bo SHI

    2005-01-01

    The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However,like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites,distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demonstrated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior observed during development and pathogenesis.

  20. Ultrasound Technologies for the Spatial Patterning of Cells and Extracellular Matrix Proteins and the Vascularization of Engineered Tissue

    Science.gov (United States)

    Garvin, Kelley A.

    Technological advancements in the field of tissue engineering could save the lives of thousands of organ transplant patients who die each year while waiting for donor organs. Currently, two of the primary challenges preventing tissue engineers from developing functional replacement tissues and organs are the need to recreate complex cell and extracellular microenvironments and to vascularize the tissue to maintain cell viability and function. Ultrasound is a form of mechanical energy that can noninvasively and nondestructively interact with tissues at the cell and protein level. In this thesis, novel ultrasound-based technologies were developed for the spatial patterning of cells and extracellular matrix proteins and the vascularization of three-dimensional engineered tissue constructs. Acoustic radiation forces associated with ultrasound standing wave fields were utilized to noninvasively control the spatial organization of cells and cell-bound extracellular matrix proteins within collagen-based engineered tissue. Additionally, ultrasound induced thermal mechanisms were exploited to site-specifically pattern various extracellular matrix collagen microstructures within a single engineered tissue construct. Finally, ultrasound standing wave field technology was used to promote the rapid and extensive vascularization of three-dimensional tissue constructs. As such, the ultrasound technologies developed in these studies have the potential to provide the field of tissue engineering with novel strategies to spatially pattern cells and extracellular matrix components and to vascularize engineered tissue, and thus, could advance the fabrication of functional replacement tissues and organs in the field of tissue engineering.

  1. Tailoring the properties of cholecyst-derived extracellular matrix using carbodiimide cross-linking.

    LENUS (Irish Health Repository)

    Burugapalli, Krishna

    2009-01-01

    Modulation of properties of extracellular matrix (ECM) based scaffolds is key for their application in the clinical setting. In the present study, cross-linking was used as a tool for tailoring the properties of cholecyst-derived extracellular matrix (CEM). CEM was cross-linked with varying cross-linking concentrations of N,N-(3-dimethyl aminopropyl)-N\\'-ethyl carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS). Shrink temperature measurements and ATR-FT-IR spectra were used to determine the degree of cross-linking. The effect of cross-linking on degradation was tested using the collagenase assay. Uniaxial tensile properties and the ability to support fibroblasts were also evaluated as a function of cross-linking. Shrink temperature increased from 59 degrees C for non-cross-linked CEM to 78 degrees C for the highest EDC cross-linking concentration, while IR peak area ratios for the free -NH(2) group at 3290 cm(-1) to that of the amide I band at 1635 cm(-1) decreased with increasing EDC cross-linking concentration. Collagenase assay demonstrated that degradation rates for CEM can be tailored. EDC concentrations 0 to 0.0033 mmol\\/mg CEM were the cross-linking concentration range in which CEM showed varied susceptibility to collagenase degradation. Furthermore, cross-linking concentrations up to 0.1 mmol EDC\\/mg CEM did not have statistically significant effect on the uniaxial tensile strength, as well as morphology, viability and proliferation of fibroblasts on CEM. In conclusion, the degradation rates of CEM can be tailored using EDC-cross-linking, while maintaining the mechanical properties and the ability of CEM to support cells.

  2. Extracellular matrix stiffness modulates VEGF calcium signaling in endothelial cells: individual cell and population analysis.

    Science.gov (United States)

    Derricks, Kelsey E; Trinkaus-Randall, Vickery; Nugent, Matthew A

    2015-09-01

    Vascular disease and its associated complications are the number one cause of death in the Western world. Both extracellular matrix stiffening and dysfunctional endothelial cells contribute to vascular disease. We examined endothelial cell calcium signaling in response to VEGF as a function of extracellular matrix stiffness. We developed a new analytical tool to analyze both population based and ind