Journal articles on the topic 'Fibronectin fibers'
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1
Christopher, R. A., A. P. Kowalczyk, and P. J. McKeown-Longo. "Localization of fibronectin matrix assembly sites on fibroblasts and endothelial cells." Journal of Cell Science 110, no. 5 (March 1, 1997): 569–81. http://dx.doi.org/10.1242/jcs.110.5.569.
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Polymerization of soluble fibronectin into extracellular matrix fibers occurs through the interaction between the amino terminus of fibronectin contained within a 70 kDa fragment and ‘matrix assembly sites’ on the cell surface. The present studies were performed to localize the ‘matrix assembly sites’ (defined by 70 kDa binding sites) on newly adherent cells and on cells containing preformed fibronectin matrix. Matrix nucleation sites on newly spread cells were visualized using Texas Red conjugated 70 kDa fragment and were found to colocalize with vinculin and substrate fibronectin fibrils. Cells plated onto vitronectin coated coverslips did not exhibit any 70 kDa binding sites although these cells were well-spread with fully developed focal adhesions. Time course studies indicated that 70 kDa binding sites could be detected on newly adherent cells within 30–40 minutes following cell plating onto fibronectin coated coverslips, prior to the reorganization of substrate fibronectin into fibrils. Similarly, exogenous fibronectin conjugated with Texas Red was also colocalized with vinculin when added to newly adherent cells. The disruption of actin filaments with cytochalasin D both prevented the expression of 70 kDa binding sites and also resulted in the loss of established 70 kDa binding sites on newly spread cells. After 3 days in culture, cells organized an extensive fibronectin matrix and 70 kDa was colocalized with two distinct types of matrix fibronectin fibers: fine linear cell-associated fibers which co-stained with the beta1 integrin and coarse extracellular fibers which did not stain for the beta1 integrin. There was also a third type of fibronectin fiber which was organized into a meshwork structure. There was no localization of either beta1 or 70 kDa to these structures. Treatment of 3-day cells with cytochalasin D resulted in the disruption of cell-matrix fibers and cell-associated 70 kDa binding sites. In contrast, the coarse extracellular matrix fibers as well as the meshwork fibers were unaffected by cytochalasin. In the presence of cytochalasin D, 70 kDa bound to sites which colocalized with the coarse extracellular matrix fibers. These data suggest that de novo assembly of fibronectin matrix occurs at sites of focal adhesion and as fibronectin polymerization proceeds, matrix nucleation sites colocalize along cell associated fibronectin fibers. At later times 70 kDa is localized to a subset of more mature fibronectin-containing fibers. These results suggest that there are at least three morphologically distinct 70 kDa binding sites on adherent cells: one which colocalizes with beta1 to focal adhesions, a second which colocalizes with beta1 and fibronectin in matrix contacts, and a third which localizes to extracellular matrix fibers.
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Ba, X., Y. Meng, Y. Huang, S. Y. Kwak, S. Ge, Y. Qin, E. DiMasi, Helga Füredi-Milhofer, N. Pernodet, and Miriam Rafailovich. "In Vitro Biomineralization Induced by Self-Assembled Extracellular Matrix Proteins." Key Engineering Materials 361-363 (November 2007): 427–30. http://dx.doi.org/10.4028/www.scientific.net/kem.361-363.427.
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Extracellular matrix (ECM) proteins play an essential role during biomineralization in bone and engineered tissues. In a previous study [1], we showed that calcite preferentially nucleated on pure elastin fibers. However, the actual cellular ECM fibers are composed of a combination of proteins, primarily collagen, fibronectin and some elastin. Here we follow the calcium carbonate- and calcium phosphate- mineralization process in vitro when these ECM proteins are combined and determine the differences between these proteins in the biomineralization process. The surface morphology and mechanical properties of the protein fibers during the early stages were probed by atomic force microscopy (AFM) and shear modulation force microscopy (SMFM). The nucleation of the mineral crystals on the protein matrices was investigated by scanning electron microscopy (SEM). Preliminary data showed that the moduli of all protein fibers increased at the early stages, with collagen having the largest increase in supersaturated calcium bicarbonate solution. In metastable calcium phosphate solutions the modulus of the mixed elastin-fibronectin fibres increased to a greater extent than the moduli of the fibers composed of the single proteins. Longer exposure in the mineral solutions led to the formation of crystals templated along the self-assembled fiber structures.
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Echtermeyer, F., P. C. Baciu, S. Saoncella, Y. Ge, and P. F. Goetinck. "Syndecan-4 core protein is sufficient for the assembly of focal adhesions and actin stress fibers." Journal of Cell Science 112, no. 20 (October 15, 1999): 3433–41. http://dx.doi.org/10.1242/jcs.112.20.3433.
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The formation of focal adhesions and actin stress fibers on fibronectin is dependent on signaling through (β)1 integrins and the heparan sulfate proteoglycan syndecan-4, and we have analyzed the requirement of the glycosaminoglycan chains of syndecan-4 during these events. Chinese hamster ovary cells with mutations in key enzymes of the glycanation process do not synthesize glycosaminoglycan chains and are unable to assemble actin stress fibers and focal contacts when cultured on fibronectin. Transfection of the mutant cells with a cDNA that encodes the core protein of chicken syndecan-4 leads to the production of unglycanated core protein. The overexpression of syndecan-4 core protein in these mutant cells increases cell spreading and is sufficient for these cells to assemble actin stress fibers and focal adhesions similar to wild-type cells seeded on fibronectin and vitronectin matrices. Syndecan-4 core protein colocalizes to focal contacts in mutant cells that have been transfected with the syndecan-4 core protein cDNA. These data indicate an essential role for the core protein of syndecan-4 in the generation of signals leading to actin stress fiber and focal contact assembly.
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Flück, Martin, Matthias Chiquet, Silvia Schmutz, Marie-Hélène Mayet-Sornay, and Dominique Desplanches. "Reloading of atrophied rat soleus muscle induces tenascin-C expression around damaged muscle fibers." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 284, no. 3 (March 1, 2003): R792—R801. http://dx.doi.org/10.1152/ajpregu.00060.2002.
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The hypothesis was tested that mechanical loading, induced by hindlimb suspension and subsequent reloading, affects expression of the basement membrane components tenascin-C and fibronectin in the belly portion of rat soleus muscle. One day of reloading, but not the previous 14 days of hindlimb suspension, led to ectopic accumulation of tenascin-C and an increase of fibronectin in the endomysium of a proportion (8 and 15%) of muscle fibers. Large increases of tenascin-C (40-fold) and fibronectin (7-fold) mRNA within 1 day of reloading indicates the involvement of pretranslational mechanisms in tenascin-C and fibronectin accumulation. The endomysial accumulation of tenascin-C was maintained up to 14 days of reloading and was strongly associated with centrally nucleated fibers. The observations demonstrate that an unaccustomed increase of rat soleus muscle loading causes modification of the basement membrane of damaged muscle fibers through ectopic endomysial expression of tenascin-C.
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Dugina, Vera, Lionel Fontao, Christine Chaponnier, Jury Vasiliev, and Giulio Gabbiani. "Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors." Journal of Cell Science 114, no. 18 (September 15, 2001): 3285–96. http://dx.doi.org/10.1242/jcs.114.18.3285.
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Transforming growth factor β (TGFβ), the most established promoter of myofibroblast differentiation, induces ED-A cellular fibronectin and α-smooth muscle actin expression in fibroblastic cells in vivo and in vitro. ED-A fibronectin exerts a permissive action for α-smooth muscle actin expression. A morphological continuity (called fibronexus), a specialized form of focal adhesion, has been described between actin stress fibers that contain α-smooth muscle actin, and extracellular fibronectin, which contains the ED-A portion, in both cultured fibroblasts and granulation tissue myofibroblasts. We have studied the development of these focal adhesions in TGFβ-treated fibroblasts using confocal laser scanning microscopy, three-dimensional image reconstruction and western blots using antibodies against focal adhesion proteins. The increase in ED-A fibronectin expression induced by TGFβ was accompanied by bundling of ED-A fibronectin fibers and their association with the terminal portion of α-smooth muscle actin-positive stress fibers. In parallel, the focal adhesion size was importantly increased, and tensin and FAK were neoexpressed in focal adhesions; moreover, vinculin and paxillin were recruited from the cytoplasmic pool into focal adhesions. We have evaluated morphometrically the length and area of focal adhesions. In addition, we have evaluated biochemically their content of associated proteins and of α-smooth muscle actin after TGFβ stimulation and on this basis suggest a new focal adhesion classification, that is, immature, mature and supermature.When TGFβ-induced α-smooth muscle actin expression was blocked by soluble recombinant ED-A fibronectin, we observed that the fragment was localised into the fibronectin network at the level of focal adhesions and that focal adhesion supermaturation was inhibited. The same effect was also exerted by the ED-A fibronectin antibody IST-9. In addition, the antagonists of actin-myosin contractility BDM and ML-7 provoked the dispersion of focal adhesions and the decrease of α-smooth muscle actin content in stress fibers of pulmonary fibroblasts, which constitutively show large focal adhesions and numerous stress fibers that contain α-smooth muscle actin. These inhibitors also decreased the incorporation of recombinant ED-A into fibronectin network. Our data indicate that a three-dimensional transcellular structure containing both ED-A fibronectin and α-smooth muscle actin plays an important role in the establishment and modulation of the myofibroblastic phenotype. The organisation of this structure is regulated by intracellularly and extracellularly originated forces.
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Morris, S. M., P. J. Stone, and G. L. Snider. "Electron microscopic study of human lung tissue after in vitro exposure to elastase." Journal of Histochemistry & Cytochemistry 41, no. 6 (June 1993): 851–66. http://dx.doi.org/10.1177/41.6.8315277.
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Much of the experimental evidence supporting the hypothesis that pulmonary emphysema results from an imbalance between elastases and anti-elastases in the lung comes from animal models. The present study was designed to examine the effects on human lung tissue of the two elastases that have been most widely used to produce these animal models. Lung tissue was exposed in vitro to human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE). Although both enzymes solubilized protein at similar rates, PPE solubilized elastin five times faster than did HNE. Ultrastructurally, HNE-exposed tissue exhibited fewer damaged elastic fibers as well as some fibers that were damaged at the edges, whereas the interior of the fiber appeared intact. Elastic fibers showing damage only at the periphery were not seen in tissue exposed to PPE. Immunocytochemical studies in which antibodies to HNE and PPE were applied to thin sections of Lowicryl-embedded tissue indicated that both of these elastases could be detected in association with elastic fibers, but only in areas of the fiber that showed morphological evidence of elastase injury. Both HNE and PPE removed fibronectin from basement membranes (as determined by loss of binding of fibronectin antibodies after exposure to elastase), but neither elastase was detected on basement membrane. Loss of epithelial cells usually accompanied elastic fiber damage by HNE but not PPE.
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Moyano, José V., Alfredo Maqueda, Benito Casanova, and Angeles Garcia-Pardo. "α4β1 Integrin/Ligand Interaction Inhibits α5β1-induced Stress Fibers and Focal Adhesions via Down-Regulation of RhoA and Induces Melanoma Cell Migration." Molecular Biology of the Cell 14, no. 9 (September 2003): 3699–715. http://dx.doi.org/10.1091/mbc.e02-10-0667.
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We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7–10 fragment. Coimmobilization of FNIII4–5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7–10, or addition of soluble FNIII4–5 to cells preattached to FNIII7–10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7–10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7–10+FNIII4–5. Furthermore, addition of alpha4beta1 ligands to FNIII7–10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.
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Litvinov, R. I. "Prospects for therapeutic use of fibronectin preparations." Kazan medical journal 67, no. 5 (September 15, 1986): 391–97. http://dx.doi.org/10.17816/kazmj70725.
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Blood plasma fibronectin is a glycoprotein with a molecular weight of 450 kD consisting of two almost identical polypeptide chains linked by disulfide bonds. The normal concentration of fibronectin in adult plasma is 0.3-0.4 g/l. Plasma fibronectin is synthesized by hepatocytes and possibly by endothelial cells. In its chemical structure and immunological properties it is similar to tissue fibronectin, which is formed mainly by connective tissue cells and is a part of extracellular matrix and collagen fibers.
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Yoneda, Atsuko, Dmitriy Ushakov, Hinke A. B. Multhaupt, and John R. Couchman. "Fibronectin Matrix Assembly Requires Distinct Contributions from Rho Kinases I and -II." Molecular Biology of the Cell 18, no. 1 (January 2007): 66–75. http://dx.doi.org/10.1091/mbc.e06-08-0684.
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Extracellular matrix is integral to tissue architecture and regulates many aspects of cell behavior. Fibronectin matrix assembly involves the actin cytoskeleton and the small GTPase RhoA, but downstream signaling is not understood. Here, down-regulation of either rho kinase isoform (ROCK I or -II) by small interfering RNA treatment blocked fibronectin matrix assembly, although the phenotypes were distinct and despite persistence of the alternate kinase. Remnant fibronectin on ROCK-deficient fibroblasts was mostly punctate and more deoxycholate soluble compared with controls. Fibronectin matrix assembly defects in ROCK-deficient cells did not result from decreased synthesis/secretion, altered fibronectin mRNA splicing, metalloproteinase activity, or α5β1 integrin dysfunction. Rescue could be effected by ROCK protein restoration or phosphomimetic myosin light chain expression. However, the effect of ROCK I deficiency on fibronectin matrix assembly was secondary to altered cell surface morphology, rich in filopodia, resulting from high GTP–Cdc42 levels. Total internal reflection microscopy revealed that a submembranous pool of myosin light chain in control cells was missing in ROCK II-deficient cells and replaced by stress fibers. Together, two rho kinases contribute to fibronectin matrix assembly in a different manner and cortical myosin II-driven contractility, but not stress fibers, may be critical in this activity.
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Sugimoto, K., S. Fujii, T. Takemasa, and K. Yamashita. "Factors inducing codistribution of marginal actin fibers and fibronectin in rat aortic endothelial cells." American Journal of Physiology-Heart and Circulatory Physiology 272, no. 5 (May 1, 1997): H2188—H2194. http://dx.doi.org/10.1152/ajpheart.1997.272.5.h2188.
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Rat endothelial cells have a unique arrangement of actin fibers running with subendothelial fibronectin along the cell margins. The present study was conducted to identify the factors that are associated with codistribution of these actin fibers and fibronectin in fetal rat aortic endothelial cells, mainly using rheological techniques. Fluorescence histochemistry revealed that the codistribution pattern was established between gestational days 14 and 15. The endothelial cells changed from polygonal to spindle shaped during this gestational period. Although the aortic length remained almost unchanged during this period of gestation, both the diameter and expansion ratio of the aorta increased, by 9 and 10%, respectively. On the other hand, the wall shear rate increased rapidly during gestational days 13-16. These results indicate that the codistribution of actin fibers and subendothelial fibronectin along the margins of endothelial cells in fetal rat aorta occurs in association with a rise in stretch and shear stress and that the endothelial cells may require this change to maintain structural integrity.
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Raoufi, Mohammad, Tamal Das, Ingmar Schoen, Viola Vogel, Dorothea Brüggemann, and Joachim P. Spatz. "Nanopore Diameters Tune Strain in Extruded Fibronectin Fibers." Nano Letters 15, no. 10 (September 29, 2015): 6357–64. http://dx.doi.org/10.1021/acs.nanolett.5b01356.
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Woods, A., and J. R. Couchman. "Protein kinase C involvement in focal adhesion formation." Journal of Cell Science 101, no. 2 (February 1, 1992): 277–90. http://dx.doi.org/10.1242/jcs.101.2.277.
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Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form. Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concentrations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin beta 1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cell-binding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.
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Cosio, F. G. "Fibronectin metabolism by human mesangial cells: effects of collagens." American Journal of Physiology-Renal Physiology 264, no. 1 (January 1, 1993): F106—F119. http://dx.doi.org/10.1152/ajprenal.1993.264.1.f106.
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In the present study we assessed whether the fibronectin (FN) metabolism of human mesangial cells (HMC) in culture is influenced by the contact of HMC with collagens type I and IV. HMC were grown on collagen gels or on collagen-coated surfaces (collagen films). FN concentrations were measured by enzyme-linked immunosorbent assay; FN synthesis was measured by metabolic labeling and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, the structure of matrix FN was examined by immunofluorescence microscopy. Compared with cells grown on plastic, HMC on collagen gels or collagen films accumulated greater amounts of FN in the cell matrix, and in these cultures, matrix FN was organized into a complex mesh of fibers. FN fiber formation was more prominent in cells adherent to collagen IV than in cells adherent to collagen I, and these fibers were observed as early as day 1 in culture. HMC adherent to plastic deposited matrix FN as patches and only occasionally as FN fibers localized to the periphery of the cell. The accumulation of FN in the matrix of HMC on collagen was not due to an increased rate of FN synthesis. In fact, HMC on collagen gels synthesized less FN than HMC on plastic. The present results indicate that the accumulation of FN in the matrix of HMC on collagen is due to the fact that this FN is less likely to be released into the supernatant than the matrix FN produced by HMC on plastic. The decreased FN synthesis demonstrated by HMC on collagen gels was associated with an overall decrease in protein synthesis but was not associated with a decrease in FN mRNA levels. Finally, FN isolated from HMC on collagen gels contained a unique 90-kDa gelatin-binding FN fragment. In conclusion, collagens have effects on the synthesis, localization, organization, and catabolism of FN produced by HMC in culture. In particular, collagen IV, the collagen normally present in the glomerular mesangium, appears to influence uniquely the organization of mesangial matrix FN.
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Okech, William, Keren M. Abberton, Julia M. Kuebel, Denise C. Hocking, and Ingrid H. Sarelius. "Extracellular matrix fibronectin mediates an endothelial cell response to shear stress via the heparin-binding, matricryptic RWRPK sequence of FNIII1H." American Journal of Physiology-Heart and Circulatory Physiology 311, no. 4 (October 1, 2016): H1063—H1071. http://dx.doi.org/10.1152/ajpheart.00126.2016.
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Endothelial cells (EC) respond to mechanical forces such as shear stress in a variety of ways, one of which is cytoskeletal realignment in the direction of flow. Our earlier studies implicated the extracellular matrix protein fibronectin in mechanosensory signaling to ECs in intact arterioles, via a signaling pathway dependent on the heparin-binding region of the first type III repeat of fibrillar fibronectin (FNIII1H). Here we test the hypothesis that FNIII1H is required for EC stress fiber realignment under flow. Human umbilical vein ECs (HUVECs) exposed to defined flow conditions were used as a well-characterized model of this stress fiber alignment response. Our results directly implicate FNIII1H in realignment of stress fibers in HUVECs and, importantly, show that the matricryptic heparin-binding RWRPK sequence located in FNIII1 is required for the response. Furthermore, we show that flow-mediated stress fiber realignment in ECs adhered via α5β1-integrin-specific ligands does not occur in the absence of FHIII1H, whereas, in contrast, αvβ3-integrin-mediated stress fiber realignment under flow does not require FNIII1H. Our findings thus indicate that there are two separate mechanosignaling pathways mediating the alignment of stress fibers after exposure of ECs to flow, one dependent on αvβ3-integrins and one dependent on FNIII1H. This study strongly supports the conclusion that the RWRPK region of FNIII1H may have broad capability as a mechanosensory signaling site.
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Zubairov, D. M., I. V. Brikman, O. D. Zinkevich, R. I. Litvinov, S. I. Ibadova, R. T. Isaeva, A. F. Harrasov, et al. "Development of the drug fibronectin and substantiation of its use to stimulate corneal healing." Kazan medical journal 70, no. 1 (February 15, 1989): 48–50. http://dx.doi.org/10.17816/kazmj99767.
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The urgency of the search for remedies accelerating corneal healing after its accidental and surgical injuries is obvious. In recent years, along with physical factors, some natural and synthetic substances, fibronectin turned out to be among the most promising therapeutic agents. It is an adhesive protein of connective tissue involved in cell attachment to fibrillar substrates, formation of collagen fibers and intercellular substance. Fibronectin and its fragments have chemotactic activity and can accelerate phagocytosis reactions by macrophages and neutrophils, acting as a non-specific opsonin against many exo- and endogenous pathological microparticles. One form of this protein, called plasma fibronectin, is present in blood and has many biological properties in common with tissue fibronectin.
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Talamás-Rohana, Patricia, and Amelia Rı́os. "Actin Stress Fibers in Entamoeba histolytica Induced by Fibronectin." Archives of Medical Research 31, no. 4 (July 2000): S131—S133. http://dx.doi.org/10.1016/s0188-4409(00)00190-9.
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Peters, D. M., and D. F. Mosher. "Localization of cell surface sites involved in fibronectin fibrillogenesis." Journal of Cell Biology 104, no. 1 (January 1, 1987): 121–30. http://dx.doi.org/10.1083/jcb.104.1.121.
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Fibronectin binding sites on cultured human fibroblasts were localized by high voltage electron microscopy using either 5- or 18-nm colloidal gold beads (Au5 or Au18) bound to intact fibronectin, the 70-kD amino-terminal fragment of fibronectin that blocks incorporation of exogenous fibronectin into extracellular matrix, or 160-180-kD fragments of fibronectin with cell adhesion and heparin-binding activities. Binding sites for Au18-fibronectin on the cell surface were localized to specific regions along the edge of the fibroblast and on retraction fibers. Au18-fibronectin complexes at these sites were initially localized in clusters that co-aligned with intracellular microfilament bundles. With longer incubations, Au18-fibronectin complexes were arranged into long fibrillar networks on the cell surface and in the extracellular space. The appearance of Au18-fibronectin in these fibrillar networks and disappearance of clusters of Au18-fibronectin suggest that Au18-fibronectin complexes are arranged into matrix at specific regions of the cell surface. Au18-70-kD fragment complexes initially had a similar distribution to Au18-fibronectin complexes. With longer incubations, Au18-70-kD fragment complexes were found in long linear arrangements on the cell surface. Double labeling experiments using Au18-70-kD fragment and Au5-160-180-kD fragments showed that the 70-kD fragment and the 160-180-kD fragments bind to different regions of the cell.
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LeBaron, R. G., J. D. Esko, A. Woods, S. Johansson, and M. Höök. "Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata." Journal of Cell Biology 106, no. 3 (March 1, 1988): 945–52. http://dx.doi.org/10.1083/jcb.106.3.945.
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We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan-deficient cells did not attach to substrate composed of a heparin-binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.
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Arregui, Carlos O., Janne Balsamo, and Jack Lilien. "Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B." Journal of Cell Biology 143, no. 3 (November 2, 1998): 861–73. http://dx.doi.org/10.1083/jcb.143.3.861.
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To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.
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Huang, Yan-Bing, Hui Zheng, Xiu-Xia Yang, Cheng-Cheng Yang, Ye Liu, and Yang Liu. "Inhibition of TGF-β2-induced migration and epithelial-mesenchymal transition in ARPE-19 by sulforaphane." International Journal of Ophthalmology 14, no. 7 (July 18, 2021): 973–80. http://dx.doi.org/10.18240/ijo.2021.07.03.
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AIM: To investigate the effects of sulforaphane (SFN) on transforming growth factor (TGF)-β2 stimulated migration and epithelial-mesenchymal transition (EMT) in ARPE-19 cells. METHODS: ARPE-19 cells were cultured in the presence or absence of SFN or TGF-β2. SFN toxicity was assessed by performing a lactate dehydrogenase assay (LDH) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays, and cell migration was evaluated by Transwell migration assay. Actin stress fiber formation in ARPE-19 cells was determined using immunofluorescence analysis. Immunoblotting analysis was used to determine fibronectin and α-smooth muscle actin expressions along with the degree of Smad and Akt phosphorylation. RESULTS: SFN inhibited ARPE-19 migration. Additionally, SFN attenuated TGF-β2-induced appearance of actin stress fibers as well as fibronectin and α-smooth muscle actin expressions in these cells. SFN also hindered the TGF-β2-stimulated phosphorylation of Smad2, Smad3, and Akt. SFN showed no cytotoxicity towards ARPE-19 cells. CONCLUSION: SFN inhibits TGF-β2-stimulated migration and EMT in ARPE-19 cells, probably by preventing the establishment of actin stress fibers and Akt and Smad2/3 signaling.
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Carter, D. H., P. Sloan, and J. E. Aaron. "Immunolocalization of collagen types I and III, tenascin, and fibronectin in intramembranous bone." Journal of Histochemistry & Cytochemistry 39, no. 5 (May 1991): 599–606. http://dx.doi.org/10.1177/39.5.1707904.
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Structural components of the organic bone matrix were located by immunohistochemical techniques in fresh-frozen sections of normal and dysplastic bone. Fine and coarse birefringent fibers were identified as separate and distinctive features in the extracellular matrix by antibodies raised against human collagen Type III. The glycoprotein tenascin was located on a proportion of the fibers in a characteristic beaded pattern, which was absent in dysplastic bone. The fibers originated in the periosteum or in the fibrous stroma of the marrow cavity and were oriented with regard to both the spatial and the lamellar organization of the bone. The disposition and composition of the fibers suggests that they form a preliminary framework on which intramembranous bone modeling proceeds, and that the specific location of tenascin on the fibers in normal developing membrane bone may be important in determining the alignment of the bone tissue. Epitopes recognized by the collagen Type I and fibronectin antibodies were demonstrated throughout the mineralized matrix, but their incorporation into the collagen "Type III" fibers was evident only outside the mineralized matrix.
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Ortiz Franyuti, Daniela, Maria Mitsi, and Viola Vogel. "Mechanical Stretching of Fibronectin Fibers Upregulates Binding of Interleukin-7." Nano Letters 18, no. 1 (December 28, 2017): 15–25. http://dx.doi.org/10.1021/acs.nanolett.7b01617.
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Seo, Bo Ri, Xingyu Chen, Lu Ling, Young Hye Song, Adrian A. Shimpi, Siyoung Choi, Jacqueline Gonzalez, et al. "Collagen microarchitecture mechanically controls myofibroblast differentiation." Proceedings of the National Academy of Sciences 117, no. 21 (May 8, 2020): 11387–98. http://dx.doi.org/10.1073/pnas.1919394117.
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Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.
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Kurisu, K., Y. Ohsaki, K. Nagata, T. Kukita, H. Yoshikawa, and T. Inai. "Immunoelectron microscopic localization of fibronectin in the smooth muscle layer of mouse small intestine." Journal of Histochemistry & Cytochemistry 35, no. 4 (April 1987): 411–17. http://dx.doi.org/10.1177/35.4.3546487.
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We studied the ultrastructural distribution of fibronectin in the smooth muscle layer of mouse small intestine with affinity-purified antibodies using the immunogold technique. Fibronectin was present over the pericellular area extending from the cell membrane to the extracellular matrix beyond the basal lamina. Distribution of the glycoprotein over the pericellular area was heterogeneous, i.e., it was localized more abundantly in the narrow space between smooth muscle cells, the gaps having a width of 60-80 nm where the two dense bands in adjacent cells matched each other. Such localization suggests that fibronectin contributes to cell adhesion. Within the basement membrane, gold label was localized both in lamina lucida and lamina densa, more densely in the latter than in the former. Fibronectin was also co-distributed with collagen fibers in the extracellular matrix. Within smooth muscle cells, gold particles were observed on rough endoplasmic reticulum and secretory vesicle-like structures. These results suggest that smooth muscle cells synthesize fibronectin and secrete it as a component of the basal lamina and extracellular matrix.
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Mott, Rosalind E., and Brian P. Helmke. "Mapping the dynamics of shear stress-induced structural changes in endothelial cells." American Journal of Physiology-Cell Physiology 293, no. 5 (November 2007): C1616—C1626. http://dx.doi.org/10.1152/ajpcell.00457.2006.
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Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.
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Samanta, Soumen, Diana Gaad, Eva Cabet, Alain Lilienbaum, Ajay Singh, Dinesh K. Aswal, and Mohamed M. Chehimi. "Flexible, Biocompatible PET Sheets: A Platform for Attachment, Proliferation and Differentiation of Eukaryotic Cells." Surfaces 4, no. 4 (December 10, 2021): 306–22. http://dx.doi.org/10.3390/surfaces4040026.
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Transparent, flexible, biaxially oriented polyethylene terephthalate (PET) sheets were modified by bioactive polymer-fibronectin top layers for the attachment of cells and growth of muscle fibers. Towards this end, PET sheets were grafted with 4-(dimethylamino)phenyl (DMA) groups from the in situ generated diazonium cation precursor. The arylated sheets served as macro-hydrogen donors for benzophenone and the growth of poly(2-hydroxy ethyl methacrylate) (PHEMA) top layer by surface-confined free radical photopolymerization. The PET-PHEMA sheets were further grafted with fibronectin (FBN) through the 1,1-carbonyldiimidazole coupling procedure. The bioactive PET-PHEMA-I-FBN was then employed as a platform for the attachment, proliferation and differentiation of eukaryotic cells which after a few days gave remarkable muscle fibers, of ~120 µm length and ~45 µm thickness. We demonstrate that PET-PHEMA yields a fast growth of cells followed by muscle fibers of excellent levels of differentiation compared to pristine PET or standard microscope glass slides. The positive effect is exacerbated by crosslinking PHEMA chains with ethylene glycol dimethacrylate at initial HEMA/EGDA concentration ratio = 9/1. This works conclusively shows that in situ generated diazonium salts provide aryl layers for the efficient UV-induced grafting of biocompatible coating that beneficially serve as platform for cell attachment and growth of muscle fibers.
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Drenckhahn, D., and J. Wagner. "Stress fibers in the splenic sinus endothelium in situ: molecular structure, relationship to the extracellular matrix, and contractility." Journal of Cell Biology 102, no. 5 (May 1, 1986): 1738–47. http://dx.doi.org/10.1083/jcb.102.5.1738.
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In the present study, we investigated structural and functional aspects of stress fibers in a cell type in situ, i.e., the sinus endothelium of the human spleen. In this cell type, stress fibers extend underneath the basal plasma membrane and are arranged parallel to the cellular long axis. Ultrastructurally, the stress fibers were found to be composed of thin actin-like filaments (5-8 nm) and thick myosin-like filaments (10-15 nm X 300 nm). Actin filaments displayed changes in polarity (determined by S-1-myosin subfragment decoration), which may allow a sliding filament mechanism. At their plasmalemmal attachment sites, actin filaments exhibited uniform polarity with the S-1-arrowhead complexes pointing away from the plasma membrane. Fluorescence microscopy showed that the stress fibers have a high affinity for phalloidin and antibodies to actin, myosin, tropomyosin, and alpha-actinin. Vinculin was confined to the cytoplasmic aspect of the plasmalemmal termination sites of stress fibers, while laminin, fibronectin, and collagens were located at the extracellular aspect of these stress fiber-membrane associations. Western blot analysis revealed polypeptide bands that contained actin, myosin, and alpha-actinin to be major components of isolated cells. Exposure of permeabilized cells to MgATP results in prominent changes in cellular shape caused by stress fiber contraction. It is concluded that the stress fibers in situ anchored to cell-to-extracellular matrix contacts can create tension that might allow the endothelium to resist the fluid shear forces of blood flow.
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Jallerat, Quentin, and Adam W. Feinberg. "Extracellular Matrix Structure and Composition in the Early Four-Chambered Embryonic Heart." Cells 9, no. 2 (January 24, 2020): 285. http://dx.doi.org/10.3390/cells9020285.
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During embryonic development, the heart undergoes complex morphogenesis from a liner tube into the four chambers consisting of ventricles, atria and valves. At the same time, the cardiomyocytes compact into a dense, aligned, and highly vascularized myocardium. The extracellular matrix (ECM) is known to play an important role in this process but understanding of the expression and organization remains incomplete. Here, we performed 3D confocal imaging of ECM in the left ventricle and whole heart of embryonic chick from stages Hamburger-Hamilton 28–35 (days 5–9) as an accessible model of heart formation. First, we observed the formation of a fibronectin-rich, capillary-like networks in the myocardium between day 5 and day 9 of development. Then, we focused on day 5 prior to vascularization to determine the relative expression of fibronectin, laminin, and collagen type IV. Cardiomyocytes were found to uniaxially align prior to vascularization and, while the epicardium contained all ECM components, laminin was reduced, and collagen type IV was largely absent. Quantification of fibronectin revealed highly aligned fibers with a mean diameter of ~500 nm and interfiber spacing of ~3 µm. These structural parameters (volume, spacing, fiber diameter, length, and orientation) provide a quantitative framework to describe the organization of the embryonic ECM.
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Toshima, Jiro, Junko Y. Toshima, Toru Amano, Neng Yang, Shuh Narumiya, and Kensaku Mizuno. "Cofilin Phosphorylation by Protein Kinase Testicular Protein Kinase 1 and Its Role in Integrin-mediated Actin Reorganization and Focal Adhesion Formation." Molecular Biology of the Cell 12, no. 4 (April 2001): 1131–45. http://dx.doi.org/10.1091/mbc.12.4.1131.
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Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.
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Partridge, C. A. "Hypoxia and reoxygenation stimulate biphasic changes in endothelial monolayer permeability." American Journal of Physiology-Lung Cellular and Molecular Physiology 269, no. 1 (July 1, 1995): L52—L58. http://dx.doi.org/10.1152/ajplung.1995.269.1.l52.
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Incubation of bovine pulmonary microvascular endothelial (BPMVE) cells in low O2 content (95% N2-5% CO2) for 4 h increased monolayer permeability to dextran almost twofold and also increased the incidence of intercellular gaps and intracellular actin stress fibers. Hypoxic incubation decreased the extracellular matrix contents of fibronectin and vitronectin, proteins that serve as anchorage points for the endothelial cells. This state was reversed after 24 h of hypoxic incubation, and the BPMVE monolayer permeability to dextran was less than that of normoxic controls. The monolayer had fewer intercellular gaps and stress fibers, and the extracellular matrix contained increased amounts of fibronectin, vitronectin, and type I collagen. These alterations stimulated by 24 h of hypoxic incubation were resolved within 4 h of reoxygenation in room air supplemented with 5% CO2. These studies indicate that incubation of endothelial monolayers in hypoxic conditions first increases and then decreases monolayer permeability, through increased and decreased formation of intercellular gaps.
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Bradshaw, Mark J., Man C. Cheung, Daniel J. Ehrlich, and Michael L. Smith. "Using Molecular Mechanics to Predict Bulk Material Properties of Fibronectin Fibers." PLoS Computational Biology 8, no. 12 (December 27, 2012): e1002845. http://dx.doi.org/10.1371/journal.pcbi.1002845.
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Wojciak-Stothard, B., M. Denyer, M. Mishra, and R. A. Brown. "Adhesion, orientation, and movement of cells cultured on ultrathin fibronectin fibers." In Vitro Cellular & Developmental Biology - Animal 33, no. 2 (February 1997): 110–17. http://dx.doi.org/10.1007/s11626-997-0031-4.
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Peleg, Orit, Thierry Savin, German V. Kolmakov, Isaac G. Salib, Anna C. Balazs, Martin Kröger, and Viola Vogel. "Fibers with Integrated Mechanochemical Switches: Minimalistic Design Principles Derived from Fibronectin." Biophysical Journal 103, no. 9 (November 2012): 1909–18. http://dx.doi.org/10.1016/j.bpj.2012.09.028.
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Tranqui, L., Y. Usson, C. Marie, and M. R. Block. "Adhesion of CHO cells to fibronectin is mediated by functionally and structurally distinct adhesion plaques." Journal of Cell Science 106, no. 1 (September 1, 1993): 377–87. http://dx.doi.org/10.1242/jcs.106.1.377.
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We have investigated the dynamics between free fibronectin receptors and clusters of them organized into adhesion plaques on CHO cells using the ability of these free integrins to be endocytosed and recycled to the plasma membrane. Indirect inhibition of the endocytic cycle by monensin resulted in the subsequent internalization of free receptors, which we followed by indirect immunostaining and confocal microscopy. Consequently, all the adhesive structures that were in equilibrium with free integrins became progressively disorganized. The cellular morphological changes were analyzed and correlated with the distribution of cell-substratum contacts viewed by confocal images obtained after immunostaining with antibodies raised against the fibronectin receptor, talin, vinculin and actin. After cell adhesion to fibronectin, blockage of the endocytic cycle induced disruption of the adhesion plaques that were mainly localized at the cell periphery, and disappearance of the stress fibers. However, the cells remained firmly attached to the substratum through focal contacts localized in the central part of the cell. These central focal contacts, but not the peripheral adhesion plaques, could form when the vesicular traffic was blocked prior to adhesion and they allowed the cells to attach and flatten onto the substratum. Whereas both adhesive structures contained the same receptors linked to talin and vinculin, the central adhesive structures were attached to a short stretch of actin but never permitted the organization of stress fibers.
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Stamatoglou, S. C., R. C. Hughes, and U. Lindahl. "Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin." Journal of Cell Biology 105, no. 5 (November 1, 1987): 2417–25. http://dx.doi.org/10.1083/jcb.105.5.2417.
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Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.
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Ferriola, P. C., and W. Stewart. "Fibronectin expression and organization in mesothelial and mesothelioma cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 271, no. 5 (November 1, 1996): L804—L812. http://dx.doi.org/10.1152/ajplung.1996.271.5.l804.
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Mesothelial cells are believed to be the progenitor cells for malignant mesothelioma, a tumor associated with exposure to asbestos and other mineral fibers. Little is known regarding fibronectin (Fn) function in mesothelial and mesothelioma cells. Fn RNA, protein levels, and localization were assessed in secondary cultures and later passages of spontaneously immortalized rat pleural mesothelial (NRM) cells and in neoplastic cell lines derived from asbestos-induced mesotheliomas. NRM cells expressed similar levels of Fn RNA regardless of passage number or cell density, as determined by Northern blotting and ribonuclease protection assays. Western blotting showed that Fn protein was both secreted by NRM cells and associated with cell lysates. Immunofluorescent confocal laser scanning microscopy demonstrated that secondary cultures of NRM cells assembled Fn into abundant homogeneous fibrillar arrays organized primarily between cells, whereas later passages of NRM cells displayed abundant but less homogeneous Fn organization. Fn RNA and protein levels in neoplastic mesothelial cells were slightly less or similar to levels in NRM cells. Organization of Fn in neoplastic cells was heterogeneous compared with secondary cultures of NRM cells, but Fn fibril formation was still apparent. F-actin microfilaments were organized in both NRM and neoplastic cells; however, actin stress fibers were maintained in neoplastic cells, whereas NRM cells displayed dense actin peripheral bands at high density. The maintenance of organized Fn and actin in mesothelioma cells is surprising and may contribute to the localized growth and invasive properties of these tumors.
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Singer, I. I., S. Scott, D. W. Kawka, D. M. Kazazis, J. Gailit, and E. Ruoslahti. "Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation." Journal of Cell Biology 106, no. 6 (June 1, 1988): 2171–82. http://dx.doi.org/10.1083/jcb.106.6.2171.
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We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin-coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate-bound vitronectin.
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Hyytiäinen, Marko, and Jorma Keski-Oja. "Latent TGF-β binding protein LTBP-2 decreases fibroblast adhesion to fibronectin." Journal of Cell Biology 163, no. 6 (December 22, 2003): 1363–74. http://dx.doi.org/10.1083/jcb.200309105.
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We have analyzed the effects of latent TGF-β binding protein 2 (LTBP-2) and its fragments on lung fibroblast adhesion. Quantitative cell adhesion assays indicated that fibroblasts do not adhere to full-length LTBP-2. Interestingly, LTBP-2 had dominant disrupting effects on the morphology of fibroblasts adhering to fibronectin (FN). Fibroblasts plated on LTBP-2 and FN substratum exhibited less adherent morphology and displayed clearly decreased actin stress fibers than cells plated on FN. These cells formed, instead, extensive membrane ruffles. LTBP-2 had no effects on cells adhering to collagen type I. Fibroblasts adhered weakly to the NH2-terminal fragment of LTBP-2. Unlike FN, this fragment did not augment actin stress fiber formation. Interestingly, the adhesion-mediating and cytoskeleton-disrupting effects were localized to the same NH2-terminal proline-rich region of LTBP-2. LTBP-2 and its antiadhesive fragment bound to FN in vitro, and the antiadhesive fragment associated with the extracellular matrix FN fibrils. These observations reveal a potentially important role for LTBP-2 as an antiadhesive matrix component.
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Spiegel, S., K. M. Yamada, B. E. Hom, J. Moss, and P. H. Fishman. "Fibrillar organization of fibronectin is expressed coordinately with cell surface gangliosides in a variant murine fibroblast." Journal of Cell Biology 102, no. 5 (May 1, 1986): 1898–906. http://dx.doi.org/10.1083/jcb.102.5.1898.
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NCTC 2071A cells, a line of transformed murine fibroblasts, grow in serum-free medium, are deficient in gangliosides, synthesize fibronectin, but do not retain and organize it on the cell surface. When the cells are exposed to exogenous gangliosides, fibrillar strands of fibronectin become attached to the cell surface. A morphologically distinct variant of NCTC 2071A cells was observed to both retain cell surface fibronectin and organize it into a fibrillar network when the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody. A revertant cell type appeared to resemble the parental NCTC 2071A cells in terms of morphology and fibronectin organization. All three cell types were subjected to mild NaIO4 oxidation and reduction with KB3H4 of very high specific radioactivity in order to label the sialic acid residues of surface gangliosides. The variant had much more surface gangliosides than the parental, particularly more complex gangliosides corresponding to GM1 and GD1a. The surface gangliosides of the revertant were intermediate between the parental and the variant. By using sialidase, which hydrolyzes GD1a to GM1, and 125I-labeled cholera toxin, which binds specifically to GM1, the identity and levels of these gangliosides were confirmed in the three cell types. When variant cells were exposed to sialidase for 2 d, there appeared to be little change in fibronectin organization. Concomitant treatment of the cells with the B subunit of cholera toxin, which bound to all the surface GM1 including that generated by the sialidase, however, eliminated the fibrillar network of fibronectin. In addition, exposure of the variant cells to a 70,000-mol-wt fragment of fibronectin, which lacks the cell attachment domain but contains a matrix assembly domain, inhibited the formation of fibers. Finally, all three cell types were assayed for their ability to attach to and spread on fibronectin-coated surfaces; no significant differences were found. Our results further establish that the ability of a cell to organize fibronectin into an extracellular matrix is dependent on certain gangliosides, but they also indicate that cell adhesion to fibronectin is independent of these gangliosides. We suggest that matrix organization and cell attachment and spreading are based on separate mechanisms and that these functions are associated with different cell surface "receptors."
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Goodman, S. L., G. Risse, and K. von der Mark. "The E8 subfragment of laminin promotes locomotion of myoblasts over extracellular matrix." Journal of Cell Biology 109, no. 2 (August 1, 1989): 799–809. http://dx.doi.org/10.1083/jcb.109.2.799.
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The locomotion of murine myoblasts over the extracellular matrix components laminin and fibronectin was analyzed using quantitative videomicroscopy, and the organization of the cytoskeleton was observed in parallel immunofluorescence studies. Cells plated on the laminin-nidogen complex locomoted twice as fast as on laminin alone. The main form of translocation on laminin was a jerky cycle of prolonged lamellipod extension followed by rapid (approximately 200- less than 500 microh h-1) movement of the cell body into the extended lamellipod. The locomotion-stimulating activity of laminin resides in the elastase digestion fragment E8, part of the laminin long arm, while the E1-4 fragment containing the three short arms is inactive. Myoblasts moved poorly over fibronectin irrespective of whether high, intermediate, or low coating concentrations were used (approximately 5,000- approximately 10 fmol cm-2). In contrast, the locomotory responses both to laminin and to E8 peaked sharply at coating concentrations approximately 20-50 fmol cm-2 and decreased at higher concentrations. This response corresponds to that expected for a haptotactic stimulant. When cells locomoted over a mixed substrate of laminin and fibronectin, the fibronectin effects appeared to predominate. The cytoskeleton has been implicated in many cellular motile processes. Within 6 h on fibronectin many cells expressed vinculin-containing focal contacts, elaborated stress fibers and had periodically organized alpha actinin, whereas on laminin, most cells showed diffuse vinculin and alpha actinin and a fine meshlike actin cytoskeleton. We conclude that the poor locomotion of cells over fibronectin is because of the cytoskeletal stabilization it induces.
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Mukhatyar, Vivek J., Manuel Salmerón-Sánchez, Soumon Rudra, Shoumit Mukhopadaya, Thomas H. Barker, Andrés J. García, and Ravi V. Bellamkonda. "Role of fibronectin in topographical guidance of neurite extension on electrospun fibers." Biomaterials 32, no. 16 (June 2011): 3958–68. http://dx.doi.org/10.1016/j.biomaterials.2011.02.015.
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Yamagata, M., S. Saga, M. Kato, M. Bernfield, and K. Kimata. "Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts. Implications for their roles in cell-substratum adhesion." Journal of Cell Science 106, no. 1 (September 1, 1993): 55–65. http://dx.doi.org/10.1242/jcs.106.1.55.
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We showed previously that a large chondroitin sulfate proteoglycan, PG-M (also known as versican), inhibits cell-substratum adhesion, while basement membrane heparan sulfate proteoglycan (recently named perlecan) does not (Yamagata et al. (1989) J. Biol. Chem. 264, 8012–8018). To extend our understanding of the adhesive function of these proteoglycans, we examined the pericellular localization of the proteoglycans and their ligands and also that of some matrix receptors and cytoskeletal molecules in various fibroblast culture systems. PG-M was abundant in the subcellular space of fibroblasts, but was excluded selectively from focal contacts where vinculin, integrins and fibronectin were localized. Hyaluronan, CD44 and tenascin were distributed similarly as PG-M. In contrast, perlecan was associated with fibronectin and was included in focal contacts. Syndecan-1, a membrane heparan sulfate/chondroitin sulfate proteoglycan, was associated with fibronectin at the cell surface, partly at focal contacts and in association with stress fibers. Thus, complexes of PG-M with hyaluronan, tenascin and CD44, are not involved in focal contacts. On the other hand, perlecan and syndecan-1 together with fibronectin may participate in focal contacts. The difference in localization between these proteoglycans may be related to their glycosaminoglycan content and to their distinctive roles in cell-substratum adhesion.
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Priddle, Helen, Lance Hemmings, Susan Monkley, Alison Woods, Bipin Patel, Deborah Sutton, Graham A. Dunn, Daniel Zicha, and David R. Critchley. "Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells." Journal of Cell Biology 142, no. 4 (August 24, 1998): 1121–33. http://dx.doi.org/10.1083/jcb.142.4.1121.
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We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.
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Taipale, J., J. Saharinen, K. Hedman, and J. Keski-Oja. "Latent transforming growth factor-beta 1 and its binding protein are components of extracellular matrix microfibrils." Journal of Histochemistry & Cytochemistry 44, no. 8 (August 1996): 875–89. http://dx.doi.org/10.1177/44.8.8756760.
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We studied the localization of latent transforming growth factor-beta 1 (TGF-beta 1) and its binding protein (LTBP-1) in the extracellular matrix of cultured human fibroblasts by immunofluorescence and immunoelectron microscopy. Immunofluorescence of confluent fibroblast cultures indicated that LTBP-1 localizes to extracellular fibrillar structures resembling fibronectin-collagen matrix. Similar fibrillar structures were detected in cells stained with antibodies specific for TGF-beta 1 propeptide (beta 1-LAP). Both LTBP-1 and beta 1-LAP colocalized with fibronectin in double immunofluorescence analysis. These fibrillar structures were resistant to extraction with sodium deoxycholate, which is further evidence that LTBP-1 and large latent TGF-beta 1 complexes are integral components of the extracellular matrix. SV-40-transformed human fibroblasts lacked extracellular LTBP-1 fibers. EM analysis revealed approximately 10-nm-thick microfibrils that were labeled by anti-LTBP at 90-140-nm intervals. In addition, LTBP-1 was found in structures that were heavily labeled for fibronectin. The accumulation of LTBP-1 in the fibronectin matrix could be reconstituted in vitro. When isolated matrix components were immobilized on nitrocellulose and incubated with fibroblast conditioned medium, LTBP-1 from the medium associated with cellular fibronectin but not with heparan or chondroitin sulfate, vitronectin, tenascin, laminin, or collagen I or IV. The association of LTBP-1 with cellular fibronectin was abolished by treatment of the medium with plasmin, which cleaves LTBP-1 and inhibits its assembly to matrix. The present results indicate that latent TGF-beta 1 complexes are components of the extracellular matrix and suggest that alterations of the pericellular matrix could result in aberrant TGF-beta signaling.
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Pattabiraman, Padmanabhan P., and Ponugoti Vasantha Rao. "Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells." American Journal of Physiology-Cell Physiology 298, no. 3 (March 2010): C749—C763. http://dx.doi.org/10.1152/ajpcell.00317.2009.
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Elevated intraocular pressure arising from impaired aqueous humor drainage through the trabecular pathway is a major risk factor for glaucoma. To understand the molecular basis for Rho GTPase-mediated resistance to aqueous humor drainage, we investigated the possible interrelationship between actomyosin contractile properties and extracellular matrix (ECM) synthesis in human trabecular meshwork (TM) cells expressing a constitutively active form of RhoA (RhoAV14). TM cells expressing RhoAV14 exhibited significant increases in fibronectin, tenascin C, laminin, α-smooth muscle actin (α-SMA) levels, and matrix assembly in association with increased actin stress fibers and myosin light-chain phosphorylation. RhoAV14-induced changes in ECM synthesis and actin cytoskeletal reorganization were mimicked by lysophosphatidic acid and TGF-β2, known to increase resistance to aqueous humor outflow and activate Rho/Rho kinase signaling. RhoAV14, lysophosphatidic acid, and TGF-β2 stimulated significant increases in Erk1/2 phosphorylation, paralleled by profound increases in fibronectin, serum response factor (SRF), and α-SMA expression. Treatment of RhoA-activated TM cells with inhibitors of Rho kinase or Erk, on the other hand, decreased fibronectin and α-SMA levels. Although suppression of SRF expression (both endogenous and RhoA, TGF-β2-stimulated) via the use of short hairpin RNA decreased α-SMA levels, fibronectin was unaffected. Conversely, fibronectin induced α-SMA expression in an SRF-dependent manner. Collectively, data on RhoA-induced changes in actomyosin contractile activity, ECM synthesis/assembly, and Erk activation, along with fibronectin-induced α-SMA expression in TM cells, reveal a potential molecular interplay between actomyosin cytoskeletal tension and ECM synthesis/assembly. This interaction could be significant for the homeostasis of aqueous humor drainage through the pressure-sensitive trabecular pathway.
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Zak, Pavel, and Karel Dedic. "Decrease of Bone Marrow Fibrosis in Hairy Cell Leukemia (HCL) after 2-CdA Therapy." Blood 104, no. 11 (November 16, 2004): 4574. http://dx.doi.org/10.1182/blood.v104.11.4574.4574.
Full textAbstract:
Abstract Different degree of bone marrow fibrosis is commonly described in patients with HCL. Hairy cells produce and assemble an insoluble matrix of fibronectin, which is responsible for the bone marrow fibrosis. Production of fibronectin is related to activity of the disease. The aim of our study was to evaluate the change of bone marrow fibrosis before and after 2-CdA therapy. Patients and methods: 18 patients, who were treated with 2-CdA and achieved complete remission in 6 months, were included in this analyses. Trephin bone marrow biopsy were obtained before therapy and after therapy in 12 and 24 moths. The 3μm thick section were stained with Gomori reticulin stain. Grading of reticulin fibrosis (scale of 0 to IV): grade 0 - reticulin fibers (RF) absent, grade I - scattered RF or foci of fine reticular network (RN), grade II - diffuse fine RN, grade III - diffuse fine RN and scattered thick fibers, grade IV - diffuse thick reticular fibers and presence of collagen fibers. Results: Before 2-CdA therapy: reticulin fibrosis was grade 0 in 0 p., I in 3p., II in 4p, III in 11p., IV in 0p.. Decrease of reticulin fibrosis in 12 months after 2-CdA was demonstrated in 51% of patients (grade 0 in 1, I in 8p., II in 5p., III in 4p., IV in 0p.), in 24 moths 77% of patients (grade 0 in 2, I in 10p., II in 4p., III in 2p., IV in 0p). Bone marrow fibrosis was increased by one grade in 3p. in 24 months after therapy. Conclusion: Successful 2-CdA therapy in patient with 2-CdA leads to significant reduction of bone marrow fibrosis.
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Baciu, P. C., S. Saoncella, S. H. Lee, F. Denhez, D. Leuthardt, and P. F. Goetinck. "Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization." Journal of Cell Science 113, no. 2 (January 15, 2000): 315–24. http://dx.doi.org/10.1242/jcs.113.2.315.
Full textAbstract:
Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.
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Resende, Victor Quinholes, Karoline Hagata Reis-Goes, Angela Carolina Finato, Débora de Fátima Almeida-Donanzam, Amanda Ribeiro dos Santos, Jonatas Perico, Barbara Casella Amorim, and James Venturini. "Combined Silymarin and Cotrimoxazole Therapy Attenuates Pulmonary Fibrosis in Experimental Paracoccidioidomycosis." Journal of Fungi 8, no. 10 (September 27, 2022): 1010. http://dx.doi.org/10.3390/jof8101010.
Full textAbstract:
Paracoccidioidomycosis (PCM), which mainly affects rural workers, is a systemic mycosis caused by the Paracoccidioides genus that induces pulmonary sequelae in most adult patients, causing serious disability and impairing their quality of life. Silymarin is herbal medicine with an effective antifibrotic activity. Considering that in PCM, antifibrotic treatment is still not available in pulmonary fibrosis, we aimed to evaluate combined silymarin and cotrimoxazole (CMX) therapy via the intratracheal route in BALB/c mice infected with P. brasiliensis yeast. After 12 weeks of treatment, the lungs were collected for the determination of fungal burden, production of OH-proline, deposition of collagen fibers, pulmonary concentrations of cytokines, and expression of fibronectin, α-SMA, MMP-2, MMP-9, and TIMP-2. Spleen cell cultures were also performed. Our results showed that infected mice treated with combined silymarin/CMX showed lower deposition of collagen fibers in the lungs and lower pulmonary concentrations of hydroxyproline than the placebo groups. Decreased levels of TGF-β1 and fibronectin and high levels of MMP-2 and IFN-γ were also observed in this group of mice. Collectively, our findings indicate that the combination of antifungal treatment with silymarin has a potent antifibrotic effect associated with an immunomodulatory effect that potentializes the antifungal immune response.
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Gonzalez-Perez, Francisco, Stefano Cobianchi, Claudia Heimann, James B. Phillips, Esther Udina, and Xavier Navarro. "Stabilization, Rolling, and Addition of Other Extracellular Matrix Proteins to Collagen Hydrogels Improve Regeneration in Chitosan Guides for Long Peripheral Nerve Gaps in Rats." Neurosurgery 80, no. 3 (February 20, 2017): 465–74. http://dx.doi.org/10.1093/neuros/nyw068.
Full textAbstract:
Abstract BACKGROUND: Autograft is still the gold standard technique for the repair of long peripheral nerve injuries. The addition of biologically active scaffolds into the lumen of conduits to mimic the endoneurium of peripheral nerves may increase the final outcome of artificial nerve devices. Furthermore, the control of the orientation of the collagen fibers may provide some longitudinal guidance architecture providing a higher level of mesoscale tissue structure. OBJECTIVE: To evaluate the regenerative capabilities of chitosan conduits enriched with extracellular matrix-based scaffolds to bridge a critical gap of 15 mm in the rat sciatic nerve. METHODS: The right sciatic nerve of female Wistar Hannover rats was repaired with chitosan tubes functionalized with extracellular matrix-based scaffolds fully hydrated or stabilized and rolled to bridge a 15 mm nerve gap. Recovery was evaluated by means of electrophysiology and algesimetry tests and histological analysis 4 months after injury. RESULTS: Stabilized constructs enhanced the success of regeneration compared with fully hydrated scaffolds. Moreover, fibronectin-enriched scaffolds increased muscle reinnervation and number of myelinated fibers compared with laminin-enriched constructs. CONCLUSION: A mixed combination of collagen and fibronectin may be a promising internal filler for neural conduits for the repair of peripheral nerve injuries, and their stabilization may increase the quality of regeneration over long gaps.
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Bloom, Laird, Kenneth C. Ingham, and Richard O. Hynes. "Fibronectin Regulates Assembly of Actin Filaments and Focal Contacts in Cultured Cells via the Heparin-binding Site in Repeat III13." Molecular Biology of the Cell 10, no. 5 (May 1999): 1521–36. http://dx.doi.org/10.1091/mbc.10.5.1521.
Full textAbstract:
Fibroblasts, when plated on the extracellular matrix protein fibronectin (FN), rapidly spread and form an organized actin cytoskeleton. This process is known to involve both the central α5β1 integrin-binding and the C-terminal heparin-binding regions of FN. We found that within the heparin-binding region, the information necessary for inducing organization of stress fibers and focal contacts was located in a 29–amino acid segment of FN type III module 13 (III13). We did not find a cytoskeleton-organizing role for repeat III14, which had previously been implicated in this process. Within III13, the same five basic amino acids known to be most important for heparin binding were also necessary for actin organization. A substrate of III13 alone was only weakly adhesive but strongly induced formation of filopodia and lamellipodia. Stress fiber formation required a combination of III13 and III7–11(which contains the integrin α5β1 recognition site), either as a single fusion protein or as separate polypeptides, and the relative amounts of the two binding sites appeared to determine whether stress fibers or filopodia and lamellipodia were the predominant actin structures formed. We propose that a balance of signals from III13 and from integrins regulates the type of actin structures assembled by the cell.