Spatial Anisotropies and Temporal Fluctuations in Extracellular Matrix Network Texture during Early Embryogenesis

PubMed Central

Loganathan, Rajprasad; Potetz, Brian R.; Rongish, Brenda J.; Little, Charles D.

2012-01-01

Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility. At the tissue level, ECM networks provide material properties required to accommodate the large-scale deformations and forces that shape amniote embryos. In other words, the primordial biomaterial from which reptilian, avian, and mammalian embryos are molded is a dynamic composite comprised of cells and ECM. Despite its central importance during early morphogenesis we know little about the intrinsic micrometer-scale surface properties of primordial ECM networks. Here we computed, using avian embryos, five textural properties of fluorescently tagged ECM networks — (a) inertia, (b) correlation, (c) uniformity, (d) homogeneity, and (e) entropy. We analyzed fibronectin and fibrillin-2 as examples of fibrous ECM constituents. Our quantitative data demonstrated differences in the surface texture between the fibronectin and fibrillin-2 network in Day 1 (gastrulating) embryos, with the fibronectin network being relatively coarse compared to the fibrillin-2 network. Stage-specific regional anisotropy in fibronectin texture was also discovered. Relatively smooth fibronectin texture was exhibited in medial regions adjoining the primitive streak (PS) compared with the fibronectin network investing the lateral plate mesoderm (LPM), at embryonic stage 5. However, the texture differences had changed by embryonic stage 6, with the LPM fibronectin network exhibiting a relatively smooth texture compared with the medial PS-oriented network. Our data identify, and partially characterize, stage-specific regional anisotropy of fibronectin texture within tissues of a warm-blooded embryo. The data suggest that changes in ECM textural properties reflect orderly time-dependent rearrangements of a primordial biomaterial. We conclude that the ECM microenvironment changes markedly in time and space during the most important period of amniote morphogenesis—as determined by fluctuating textural properties. PMID:22693609

Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

PubMed

Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

2015-01-01

An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces.

Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma

PubMed Central

Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant’Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

2016-01-01

Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

Manganese inhibits the ability of astrocytes to promote neuronal differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giordano, Gennaro; Pizzurro, Daniella; VanDeMark, Kathryn

Manganese (Mn) is a known neurotoxicant and developmental neurotoxicant. As Mn has been shown to accumulate in astrocytes, we sought to investigate whether Mn would alter astrocyte-neuronal interactions, specifically the ability of astrocytes to promote differentiation of neurons. We found that exposure of rat cortical astrocytes to Mn (50-500 {mu}M) impaired their ability to promote axonal and neurite outgrowth in hippocampal neurons. This effect of Mn appeared to be mediated by oxidative stress, as it was reversed by antioxidants (melatonin and PBN) and by increasing glutathione levels, while it was potentiated by glutathione depletion in astrocytes. As the extracellular matrixmore » protein fibronectin plays an important role in astrocyte-mediated neuronal neurite outgrowth, we also investigated the effect of Mn on fibronectin. Mn caused a concentration-dependent decrease of fibronectin protein and mRNA in astrocytes lysate and of fibronectin protein in astrocyte medium; these effects were also antagonized by antioxidants. Exposure of astrocytes to two oxidants, H{sub 2}O{sub 2} and DMNQ, similarly impaired their neuritogenic action, and led to a decreased expression of fibronectin. Mn had no inhibitory effect on neurite outgrowth when applied directly onto hippocampal neurons, where it actually caused a small increase in neuritogenesis. These results indicate that Mn, by targeting astrocytes, affects their ability to promote neuronal differentiation by a mechanism which is likely to involve oxidative stress.« less

Cellular fibronectin response to supervised moderate aerobic training in patients with type 2 diabetes

PubMed Central

Alghadir, Ahmad H.; Gabr, Sami A.; Al-Eisa, Einas

2016-01-01

[Purpose] Physical activity is one of the most pivotal targets for the prevention and management of vascular complications, especially endothelial dysfunctions. Cellular fibronectin is an endothelium-derived protein involved in subendothelial matrix assembly. Its plasma levels reflect matrix alterations and vessel wall destruction in patients with type II diabetes. This study investigated the influence of 12 weeks of supervised aerobic training on cellular fibronectin and its relationship with insulin resistance and body weight in type II diabetic subjects. [Subjects and Methods] This study included 50 men with type II diabetes who had a mean age of 48.8 ± 14.6 years and were randomly divided into two groups: an aerobic exercise group (12 weeks, three 50 minutes sessions per week) and control group. To examine changes in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile, 5 ml of blood was taken from the brachial vein of patients before and 48 hours after completion of the exercise period and after 12 hours of fasting at rest. Data analysis was performed using the SPSS-16 software with the independent and paired t-tests. [Results] A significant decrease was observed in body mass index and body fat percentage in the experimental group. Compared with the control group, the aerobic exercise group showed a significant decrease in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile after 12 weeks of aerobic exercise. The change in cellular fibronectin showed positive significant correlation with body mass index, diabetic biomarkers, and physical activity level. [Conclusion] The results showed that supervised aerobic exercise as a stimulus can change the levels of cellular fibronectin as matrix metalloproteinase protein a long with improvement of insulin sensitivity and glycosylated hemoglobin in order to prevent cardiovascular diseases in men with diabetes PMID:27190433

[The influence of immobilized fibronectin on karyotypic variability of two rat kangaroo kidney cell lines].

PubMed

Polianskaia, G G; Goriachaia, T S; Pinaev, G P

2007-01-01

The numerical and structural karyotypic variability has been investigated in "markerless" Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 when cultivating on a fibronectin-coated surface. In cell line NBL-3-17, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due preference adhesion of cells with lower chromosome number, disturbances of mitotic apparatus and selection of SVK, which are more adopted to changes in culture conditions. Detachment of cells from the fibronectin-coated surface, followed by 5 days cultivation on a hydrophilic surface restored control distribution. In cell line NBL-3-11, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of numerical karyotypic variability did not change compared to control variants. In cell line NBL-3-17 the frequency of chromosomal aberrations under cultivation on the fibronectin-coated surface for 1, 2, 4 and 8 days did not change relative to control variants. In cell line NBL-3-11 the frequency of chromosomal aberrations under the same conditions significantly increases, mainly at the expence of chromosomal, chromatid breaks and dicentrics (telomeric association) relative to control variants. We discuss possible reasons of differences in the character of numerical and structural karyotypic variability between cell lines NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) under cultivation on fibronectin. The reasons of the observed interline karyotypic differences possibly consist in peculiarity of karyotypic structure of cell line NBL-3-11 and in the change of gene expression, namely in a dose of certain functioning genes in the hypotryploid cell line NBL-3-17.

Avian pathogenic Escherichia coli bind fibronectin and laminin.

PubMed

Ramírez, Rosa María; Almanza, Yolanda; González, Rafael; García, Santos; Heredia, Norma

2009-04-01

Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.

The predictive value of quantitative fibronectin testing in combination with cervical length measurement in symptomatic women.

PubMed

Bruijn, Merel M C; Kamphuis, Esme I; Hoesli, Irene M; Martinez de Tejada, Begoña; Loccufier, Anne R; Kühnert, Maritta; Helmer, Hanns; Franz, Marie; Porath, Martina M; Oudijk, Martijn A; Jacquemyn, Yves; Schulzke, Sven M; Vetter, Grit; Hoste, Griet; Vis, Jolande Y; Kok, Marjolein; Mol, Ben W J; van Baaren, Gert-Jan

2016-12-01

The combination of the qualitative fetal fibronectin test and cervical length measurement has a high negative predictive value for preterm birth within 7 days; however, positive prediction is poor. A new bedside quantitative fetal fibronectin test showed potential additional value over the conventional qualitative test, but there is limited evidence on the combination with cervical length measurement. The purpose of this study was to compare quantitative fetal fibronectin and qualitative fetal fibronectin testing in the prediction of spontaneous preterm birth within 7 days in symptomatic women who undergo cervical length measurement. We performed a European multicenter cohort study in 10 perinatal centers in 5 countries. Women between 24 and 34 weeks of gestation with signs of active labor and intact membranes underwent quantitative fibronectin testing and cervical length measurement. We assessed the risk of preterm birth within 7 days in predefined strata based on fibronectin concentration and cervical length. Of 455 women who were included in the study, 48 women (11%) delivered within 7 days. A combination of cervical length and qualitative fibronectin resulted in the identification of 246 women who were at low risk: 164 women with a cervix between 15 and 30 mm and a negative fibronectin test (<50 ng/mL; preterm birth rate, 2%) and 82 women with a cervix at >30 mm (preterm birth rate, 2%). Use of quantitative fibronectin alone resulted in a predicted risk of preterm birth within 7 days that ranged from 2% in the group with the lowest fibronectin level (<10 ng/mL) to 38% in the group with the highest fibronectin level (>500 ng/mL), with similar accuracy as that of the combination of cervical length and qualitative fibronectin. Combining cervical length and quantitative fibronectin resulted in the identification of an additional 19 women at low risk (preterm birth rate, 5%), using a threshold of 10 ng/mL in women with a cervix at <15 mm, and 6 women at high risk (preterm birth rate, 33%) using a threshold of >500 ng/mL in women with a cervix at >30 mm. In women with threatened preterm birth, quantitative fibronectin testing alone performs equal to the combination of cervical length and qualitative fibronectin. Possibly, the combination of quantitative fibronectin testing and cervical length increases this predictive capacity. Cost-effectiveness analysis and the availability of these tests in a local setting should determine the final choice. Copyright © 2016 Elsevier Inc. All rights reserved.

SPR imaging biosensor for the quantitation of fibronectin concentration in blood samples.

PubMed

Sankiewicz, Anna; Romanowicz, Lech; Pyc, Marlena; Hermanowicz, Adam; Gorodkiewicz, Ewa

2018-02-20

The purpose of this study was presentation of a new biosensor capable of determination of fibronectin. This biosensor was based on the specific interaction of anti-fibronectin antibody produced in rabbit with fibronectin. The surface plasmon resonance imaging (SPRI) technique was used as a detecting method. Optimization and characterization properties of the biosensor were studied. The determination of fibronectin concentration in natural samples was done. The results were compared with a reference method (Enzyme-Linked Immunosorbent Assay-ELISA). The analytically useful dynamic response range of biosensor is between 5 and 400ngmL -1 . The detection limit is 1.5ngmL -1 and limit quantification is 5ngmL -1 . The proposed SPRI biosensor showed good selectivity for potential interferences. It was applied to determine fibronectin concentrations in plasma of healthy donors and of patients after thermal injury. Good correlations between results obtained using the SPRI biosensor and ELISA test (correlation coefficients for healthy donors 0.996, for patients 0.984) were obtained. The average fibronectin concentration of healthy donors was 140.5±24.6μgmL -1 and the average fibronectin concentration of patients was 601.5±72.1μgmL -1 , which was in agreement with results obtained by other investigators. The obtained results indicate that the developed biosensor may be a candidate for monitoring fibronectin concentration in blood samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix

PubMed Central

1989-01-01

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern- regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM. PMID:2473081

Gap junction communications influence upon fibroblast synthesis of Type I collagen and fibronectin.

PubMed

Ehrlich, H Paul; Sun, Bonnie; Saggers, Gregory C; Kromath, Fatuma

2006-07-01

In rats polyvinyl alcohol sponge subcutaneous implants treated with gap junctional intercellular communications (GJIC) uncouplers showed reduced deposition of connective tissue. Do uncouplers inhibit the synthesis and deposition of a new connective tissue by fibroblasts? Confluent human dermal fibroblasts in serum-free medium received either endosulfan or oleamide, GJIC uncouplers. Collected media were subjected to Dot Blot analysis for native Type I collagen and fibronectin. Uncoupler-treated fibroblasts released less Type I collagen, while there was no change in fibronectin release. Collagen synthesis was restored to normal, when the uncouplers were removed, showing that these uncouplers were reversible and not toxic to cells. Northern blot analysis revealed procollagen alpha1 (I) mRNA was minimally affected by endosulfan. Oleamide-treated 17-day chick embryo calvaria explants were incubated with Type I collagen antibody, frozen, cryosectioned, and then subjected to rhodamine (Rh) tagged anti-mouse-IgG antibody, to detect newly deposited Type I collagen. Fluorescent antibody-collagen complexes were localized on the periphery of cells in control calvaria, but absent around cells in oleamide-treated calvaria. GJIC optimize collagen synthesis but not fibronectin synthesis. The lack of connective tissue deposited in granulation tissues treated with uncouplers appears related to the inhibition of collagen synthesis. These findings suggest that altering GJIC might control collagen deposition in scarring. 2006 Wiley-Liss, Inc.

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine.

PubMed

Uhlenkott, C E; Huijzer, J C; Cardeiro, D J; Elstad, C A; Meadows, G G

1996-03-01

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

Epithelial-mesenchymal status influences how cells deposit fibrillin microfibrils.

PubMed

Baldwin, Andrew K; Cain, Stuart A; Lennon, Rachel; Godwin, Alan; Merry, Catherine L R; Kielty, Cay M

2014-01-01

Here, we show that epithelial-mesenchymal status influences how cells deposit extracellular matrix. Retinal pigmented epithelial (RPE) cells that expressed high levels of E-cadherin and had cell-cell junctions rich in zona occludens (ZO)-1, β-catenin and heparan sulfate, required syndecan-4 but not fibronectin or protein kinase C α (PKCα) to assemble extracellular matrix (fibrillin microfibrils and perlecan). In contrast, RPE cells that strongly expressed mesenchymal smooth muscle α-actin but little ZO-1 or E-cadherin, required fibronectin (like fibroblasts) and PKCα, but not syndecan-4. Integrins α5β1 and/or α8β1 and actomyosin tension were common requirements for microfibril deposition, as was heparan sulfate biosynthesis. TGFβ, which stimulates epithelial-mesenchymal transition, altered gene expression and overcame the dependency on syndecan-4 for microfibril deposition in epithelial RPE cells, whereas blocking cadherin interactions disrupted microfibril deposition. Renal podocytes had a transitional phenotype with pericellular β-catenin but little ZO-1; they required syndecan-4 and fibronectin for efficient microfibril deposition. Thus, epithelial-mesenchymal status modulates microfibril deposition.

Cell-micropatterning by micromolding in capillary technique based on UV polymerization

NASA Astrophysics Data System (ADS)

Park, Min J.; Choi, Won M.; Park, O. O.

2006-01-01

Although optical lithography or photolithography is one of the most well-established techniques for micro, nano-fabrication, its usage with proteins and cells is restricted by steps that must be carried out in harsh organic solvents. Here, we present simple methods for cell-micropatterning using poly(dimethylsiloxane) (PDMS) as a mold. Cell non-adhesive surface or nonfouling surface providing a physico-chemical barrier to cell attachment was introduced for biomaterial pattering, where cells fail to interact with the surface over desired periods of time determined by each application. Poly(ethylene glycol) (PEG) was selected as nonfouling material to inhibit protein adsorption from biological media. The fouling resistance of PEG polymer is often explained by a steric repulsion interaction, resulting from the compression of PEG chains as proteins approach the surface. We also chose fibronectin to direct cell attachment because it is an extracellular matrix protein that is involved in the adhesion and spreading of anchorage-dependent cells. In our experiment, we propose two methods by application of micromolding in capillary (MIMIC) method based on UV polymerization to obtain a surface of alternating PEG and fibronectin. First to fabricate PEG microstructure via MIMIC method, a pre-patterned PDMS mold is placed on a desired substrate, and then the relief structure in the mold forms a network of empty channels. A drop of ethylene glycol monomer solution containing initiator for UV polymerization is placed at the open ends of the network of channels, which is then polymerized by exposure to UV light at room temperature. Once PEG microstructure is fabricated, incubation of the patterned surface in a fibronectin-containing solution allows back-filling of only the bare regions with fibronectin via adsorption. In the alternative method, a substrate is first incubated in a fibronectin-containing solution, leading to the adsorption of fibronectin over the entire surface, and the fibronectin-adsorbed substrate is then micropatterned with the PEG by MIMIC based on UV polymerization. Both methods create reproducible alternating PEG and fibronectin patterns applicable to cell-surface interactions on the microscale.

Loss of an actin crosslinker uncouples cell spreading from cell stiffening on gels with a gradient of stiffness

NASA Astrophysics Data System (ADS)

Wen, Qi; Byfield, Fitzroy J.; Nordstrom, Kerstin; Arratia, Paulo E.; Miller, R. Tyler; Janmey, Paul A.

2009-03-01

We use microfluidics techniques to produce gels with a gradient of stiffness to show the essential function of the actin crosslinker filamin A in cell responses to mechanical stimuli. M2 melanoma cells null for filamin A do not alter their adherent area in response to increased substrate stiffness when they link to the substrate only through collagen receptors, but change adherent area normally when bound through fibronectin receptors. In contrast, filamin A-replete A7 cells change adherent area on both substrates and respond more strongly to collagen 1-coated gels than to fibronectin-coated gels. A7 cells alter their stiffness, as measured by atomic force microscopy, to match the elastic modulus of the substrate immediately adjacent to them on the gradient. M2 cells, in contrast, maintain a constant stiffness on all substrates that is as low as that of A7 cells on the softest gels achievable (1000 Pa). By contrasting the responses of these cell types to different adhesive substrates, cell spreading can be dissociated from stiffening.

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin.

PubMed

Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d' El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington L C

2015-08-07

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55-89] μm(2) for uninfected and 41 [34-51] μm(2) for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm(2) s(-1) ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm(2) s(-1) ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.

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

PubMed Central

Shi, Feng; Sottile, Jane

2011-01-01

The extracellular matrix (ECM) is dynamically remodeled by cells during development, normal tissue homeostasis and in a variety of disease processes. We previously showed that fibronectin is an important regulator of ECM remodeling. The deposition and/or polymerization of fibronectin into the ECM controls the deposition and stability of other ECM molecules. In addition, agents that inhibit fibronectin polymerization promote the turnover of fibronectin fibrils and enhance ECM fibronectin endocytosis and intracellular degradation. Endocytosis of ECM fibronectin is regulated by β1 integrins, including α5β1 integrin. We have examined the role of extracellular proteases in regulating ECM fibronectin turnover. Our data show that membrane type matrix metalloproteinase 1 (MT1-MMP; also known as MMP14) is a crucial regulator of fibronectin turnover. Cells lacking MT1-MMP show reduced turnover and endocytosis of ECM fibronectin. MT1-MMP regulates ECM fibronectin remodeling by promoting extracellular cleavage of fibronectin and by regulating α5β1-integrin endocytosis. Our data also show that fibronectin polymerization stabilizes fibronectin fibrils and inhibits ECM fibronectin endocytosis by inhibiting α5β1-integrin endocytosis. These data are the first to show that an ECM protein and its modifying enzyme can regulate integrin endocytosis. These data also show that integrin trafficking plays a major role in modulating ECM fibronectin remodeling. The dual dependence of ECM fibronectin turnover on extracellular proteolysis and endocytosis highlights the complex regulatory mechanisms that control ECM remodeling to ensure maintenance of proper tissue function. PMID:22159414

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

PubMed Central

Little, William C.; Smith, Michael L.; Ebneter, Urs; Vogel, Viola

2013-01-01

In response to growing needs for quantitative biochemical and cellular assays that address whether the extracellular matrix (ECM) acts as a mechanochemical signal converter to co-regulate cellular mechanotransduction processes, a new assay is presented where plasma fibronectin fibers are manually deposited onto elastic sheets, while force-induced changes in protein conformation are monitored by fluorescence resonance energy transfer (FRET). Fully relaxed assay fibers can be stretched at least 5–6 fold, which involves Fn domain unfolding, before the fibers break. In native fibroblast ECM, this full range of stretch-regulated conformations coexists in every field of view confirming that the assay fibers are physiologically relevant model systems. Since alterations of protein function will directly correlate with their extension in response to force, the FRET vs. strain curves presented herein enable the mapping of fibronectin strain distributions in 2D and 3D cell cultures with high spatial resolution. Finally, cryptic sites for fibronectin’s N-terminal 70-kD fragment were found to be exposed at relatively low strain, demonstrating the assay’s potential to analyze stretch-regulated protein-rotein interactions. PMID:18417335

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

PubMed Central

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

2016-01-01

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. PMID:27264108

Biosynthesis of fibronectin by rabbit aorta.

PubMed

Takasaki, I; Chobanian, A V; Brecher, P

1991-09-15

The in vitro interactions between vascular cells and fibronectin have been shown to influence phenotypic expression of both cultured endothelial and smooth muscle cells. To more effectively assess the potential functional role of fibronectin in vivo in modulating vascular phenotypes, we have established methodology for studying fibronectin biosynthesis in the rabbit aorta using aortic rings that are morphologically and functionally intact and metabolically active. Aortic rings were incubated with 35S-labeled methionine in a supplemented physiological salt solution. The tissue was fractionated, and quantitative immunoprecipitation was performed using a polyclonal antibody directed against human plasma fibronectin. Newly synthesized fibronectin was most abundant in the fraction solubilized using 4% sodium dodecyl sulfate and in the incubation medium. In all fractions studied, fibronectin was present predominantly as a dimer with no detectable aggregates of fibronectin. Pulse-chase experiments showed that a substantial amount of newly synthesized fibronectin was found in the 4% sodium dodecyl sulfate extract after only 1 h, suggesting that fibronectin was rapidly incorporated into the extracellular matrix. The more soluble forms of newly synthesized fibronectin appeared to be the precursors for secreted fibronectin, and no precursor-product relationship between soluble and insoluble fibronectin was found. Dissection of aortic rings following incubation with labeled methionine showed that newly synthesized fibronectin was uniformally distributed in both intima-media and media-adventitia segments. Endothelial cell denudation caused only a 20% decrease of fibronectin biosynthesis concomitant with similar changes in total protein biosynthesis, consistent with the medial smooth muscle cell as the major source of newly synthesized fibronectin. Biosynthesis of fibronectin was increased following a 24-h preincubation of the aortic rings, and concomitant increases in steady state mRNA for fibronectin were found. These in vitro studies documented the utility of aortic rings for the general purpose of studying protein synthesis in vascular cells and provide new information on the characteristics of fibronectin biosynthesis by aortic tissue.

Fibronectin is a survival factor for differentiated osteoblasts

NASA Technical Reports Server (NTRS)

Globus, R. K.; Doty, S. B.; Lull, J. C.; Holmuhamedov, E.; Humphries, M. J.; Damsky, C. H.

1998-01-01

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.

Evidence for the evolution of tenascin and fibronectin early in the chordate lineage.

PubMed

Tucker, Richard P; Chiquet-Ehrismann, Ruth

2009-02-01

Fibronectin and tenascin are extracellular matrix glycoproteins that play important roles in cell adhesion and motility. In a previous study we provided evidence that tenascin first appeared early in the chordate lineage. As tenascin has been proposed to act, in part, through modulation of cell-fibronectin interactions, we sought here to identify fibronectin genes in non-vertebrate chordates and other invertebrates to determine if tenascin and fibronectin evolved separately or together, and to identify phylogenetically conserved features of both proteins. We found that the genome of the urochordate Ciona savignyi contains both a tenascin gene and a gene encoding a fibronectin-like protein with fibronectin type 1, 2 and 3 repeats. The genome of the cephalochordate Branchiostoma floridae (amphioxus) also has a tenascin gene. However, we could not identify a fibronectin-like gene in B. floridae, nor could we identify fibronectin or tenascin genes in echinoderms, protostomes or cnidarians. If urochordates are more closely related to vertebrates, tenascin may have evolved before fibronectin in an ancestor common to tunicates and amphioxus. Alternatively, tenascin and fibronectin may have evolved in an ancestor common to B. floridae and C. savignyi and the fibronectin gene was subsequently lost in the cephalochordate lineage. The fibronectin-like gene from C. savignyi does not encode the RGD motif for integrin binding found in all vertebrate fibronectins, and it lacks most of the fibronectin type 1 domains believed to be critical for fibrillogenesis. In contrast, the tenascin gene in B. floridae encodes multiple RGD motifs, suggesting that integrin binding is fundamental to tenascin function.

Experiments with osteoblasts cultured under hypergravity conditions

NASA Technical Reports Server (NTRS)

Kacena, Melissa A.; Todd, Paul; Gerstenfeld, Louis C.; Landis, William J.

2004-01-01

To understand further the role of gravity in osteoblast attachment, osteoblasts were subjected to hypergravity conditions in vitro. Scanning electron microscopy of all confluent coverslips from FPA units show that the number of attached osteoblasts was similar among gravitational levels and growth durations (90 cells/microscopic field). Specifically, confluent 1.0 G control cultures contained an average of 91 +/- 8 cells/field, 3.3 G samples had 88 +/- 8 cells/field, and 4.0 G cultures averaged 90 +/- 7 cells/field. The sparsely plated cultures assessed by immunohistochemistry also had similar numbers of cells at each time point (l.0 G was similar to 3.3 and 4.0 G), but cell number changed from one time point to the next as those cells proliferated. Immunohistochemistry of centrifuged samples showed an increase in number (up to 160% increase) and thickness (up to 49% increase) of actin fibers, a decrease in intensity of fibronectin fluorescence (18-23% decrease) and an increase in number of vinculin bulbs (202-374% increase in number of vinculin bulbs/area). While hypergravity exposure did not alter the number of attached osteoblasts, it did result in altered actin, fibronectin, and vinculin elements, changing some aspects of osteoblast- substrate adhesion.

Control of T lymphocyte morphology by the GTPase Rho

NASA Technical Reports Server (NTRS)

Woodside, Darren G.; Wooten, David K.; Teague, T. Kent; Miyamoto, Yuko J.; Caudell, Eva G.; Udagawa, Taturo; Andruss, Bernard F.; McIntyre, Bradley W.

2003-01-01

BACKGROUND: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton. RESULTS: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions. CONCLUSIONS: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin

PubMed Central

Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d’ El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington LC

2015-01-01

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55–89] μm2 for uninfected and 41 [34–51] μm2 for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm2 s−1 ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm2 s−1 ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis. PMID:26249106

Endothelium-derived fibronectin regulates neonatal vascular morphogenesis in an autocrine fashion.

PubMed

Turner, Christopher J; Badu-Nkansah, Kwabena; Hynes, Richard O

2017-11-01

Fibronectin containing alternatively spliced EIIIA and EIIIB domains is largely absent from mature quiescent vessels in adults, but is highly expressed around blood vessels during developmental and pathological angiogenesis. The precise functions of fibronectin and its splice variants during developmental angiogenesis however remain unclear due to the presence of cardiac, somitic, mesodermal and neural defects in existing global fibronectin KO mouse models. Using a rare family of surviving EIIIA EIIIB double KO mice, as well as inducible endothelial-specific fibronectin-deficient mutant mice, we show that vascular development in the neonatal retina is regulated in an autocrine manner by endothelium-derived fibronectin, and requires both EIIIA and EIIIB domains and the RGD-binding α5 and αv integrins for its function. Exogenous sources of fibronectin do not fully substitute for the autocrine function of endothelial fibronectin, demonstrating that fibronectins from different sources contribute differentially to specific aspects of angiogenesis.

Development of siRNA Technology to Prevent Scar Formation in Tendon Repair

DTIC Science & Technology

2013-12-01

Anti-sense RNA technologies: Under normal conditions cells produce small interfering (si) RNAs that inhibit protein synthesis and stimulate...stimulation of fibroblast proliferation and migration, collagen and fibronectin synthesis , and altered tissue remodeling through regulation of MMPs...expression by an antisense oligonucleotide protects mice from fulminant hepatitis. Nat Biotechnol 2000;18:862-7. 7. Guha M, Xu ZG, Tung D, Lanting L

Development of siRNA Technology to Prevent Scar Formation in Tendon Repair

DTIC Science & Technology

2012-10-01

that inhibit protein synthesis and stimulate catabolism of target RNAs 1,2. This general concept can be harnessed experimentally and therapeutically...numerous mechanisms, including stimulation of fibroblast proliferation and migration, collagen and fibronectin synthesis , and altered tissue remodeling...antisense oligonucleotide protects mice from fulminant hepatitis. Nat Biotechnol 2000;18:862-7. 7. Guha M, Xu ZG, Tung D, Lanting L, Natarajan R

Fibronectin tetrapeptide is target for syphilis spirochete cytadherence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomas, D.D.; Baseman, J.B.; Alderete, J.F.

1985-11-01

The syphilis bacterium, Treponema pallidum, parasitizes host cells through recognition of fibronectin (Fn) on cell surfaces. The active site of the Fn molecule has been identified as a four-amino acid sequence, arg-gly-asp-ser (RGDS), located on each monomer of the cell-binding domain. The synthetic heptapeptide gly-arg-gly-asp-ser-pro-cys (GRGDSPC), with the active site sequence RGDS, specifically competed with SVI-labeled cell-binding domain acquisition by T. pallidum. Additionally, the same heptapeptide with the RGDS sequence diminished treponemal attachment to HEp-2 and HT1080 cell monolayers. Related heptapeptides altered in one key amino acid within the RGDS sequence failed to inhibit Fn cell-binding domain acquisition or parasitismmore » of host cells by T. pallidum. The data support the view that T. pallidum cytadherence of host cells is through recognition of the RGDS sequence also important for eukaryotic cell-Fn binding.« less

Production of Fibronectin by the Human Alveolar Macrophage: Mechanism for the Recruitment of Fibroblasts to Sites of Tissue Injury in Interstitial Lung Diseases

NASA Astrophysics Data System (ADS)

Rennard, Stephen I.; Hunninghake, Gary W.; Bitterman, Peter B.; Crystal, Ronald G.

1981-11-01

Because cells of the mononuclear phagocyte system are known to produce fibronectin and because alveolar macrophages are activated in many interstitial lung diseases, the present study was designed to evaluate a role for the alveolar macrophage as a source of the increased levels of fibronectin found in the lower respiratory tract in interstitial lung diseases and to determine if such fibronectin might contribute to the development of the fibrosis found in these disorders by being a chemoattractant for human lung fibroblasts. Production of fibronectin by human alveolar macrophages obtained by bronchoalveolar lavage and maintained in short-term culture in serum-free conditions was demonstrated; de novo synthesis was confirmed by the incorporation of [14C]proline. This fibronectin had a monomer molecular weight of 220,000 and was antigenically similar to plasma fibronectin. Macrophages from patients with idiopathic pulmonary fibrosis produced fibronectin at a rate 20 times higher than did normal macrophages; macrophages from patients with pulmonary sarcoidosis produced fibronectin at 10 times the normal rate. Macrophages from 6 of 10 patients with various other interstitial disorders produced fibronectin at rates greater than the rate of highest normal control. Human alveolar macrophage fibronectin was chemotactic for human lung fibroblasts, suggesting a functional role for this fibronectin in the derangement of the alveolar structures that is characteristic of these disorders.

Silibinin inhibits fibronectin induced motility, invasiveness and survival in human prostate carcinoma PC3 cells via targeting integrin signaling

PubMed Central

Deep, Gagan; Kumar, Rahul; Jain, Anil K; Agarwal, Chapla; Agarwal, Rajesh

2014-01-01

Prostate cancer (PCA) is the 2nd leading cause of cancer-related deaths among men in the United States. Preventing or inhibiting metastasis-related events through non-toxic agents could be a useful approach for lowering high mortality among PCA patients. We have earlier reported that natural flavonoid silibinin possesses strong anti-metastatic efficacy against PCA however, mechanism/s of its action still remains largely unknown. One of the major events during metastasis is the replacement of cell-cell interaction with integrins-based cell-matrix interaction that controls motility, invasiveness and survival of cancer cells. Accordingly, here we examined silibinin effect on advanced human PCA PC3 cells' interaction with extracellular matrix component fibronectin. Silibinin (50-200 μM) treatment significantly decreased the fibronectin (5 μg/ml)-induced motile morphology via targeting actin cytoskeleton organization in PC3 cells. Silibinin also decreased the fibronectin-induced cell proliferation and motility but significantly increased cell death in PC3 cells. Silibinin also inhibited the PC3 cells invasiveness in Transwell invasion assays with fibronectin or cancer associated fibroblasts (CAFs) serving as chemoattractant. Importantly, PC3-luc cells cultured on fibronectin showed rapid dissemination and localized in lungs following tail vein injection in athymic male nude mice; however, in silibinin-treated PC3-luc cells, dissemination and lung localization was largely compromised. Molecular analyses revealed that silibinin treatment modulated the fibronectin-induced expression of integrins (α5, αV, β1 and β3), actin-remodeling (FAK, Src, GTPases, ARP2 and cortactin), apoptosis (cPARP and cleaved caspase 3), EMT (E-cadherin and β-catenin), and cell survival (survivin and Akt) related signaling molecules in PC3 cells. Furthermore, PC3-xenograft tissue analyses confirmed the inhibitory effect of silibinin on fibronectin and integrins expression. Together, these results showed that silibinin targets PCA cells' interaction with fibronectin and inhibits their motility, invasiveness and survival; thus further supporting silibinin use in PCA intervention including its metastatic progression. PMID:25285031

Silibinin inhibits fibronectin induced motility, invasiveness and survival in human prostate carcinoma PC3 cells via targeting integrin signaling.

PubMed

Deep, Gagan; Kumar, Rahul; Jain, Anil K; Agarwal, Chapla; Agarwal, Rajesh

2014-10-01

Prostate cancer (PCA) is the 2nd leading cause of cancer-related deaths among men in the United States. Preventing or inhibiting metastasis-related events through non-toxic agents could be a useful approach for lowering high mortality among PCA patients. We have earlier reported that natural flavonoid silibinin possesses strong anti-metastatic efficacy against PCA however, mechanism/s of its action still remains largely unknown. One of the major events during metastasis is the replacement of cell-cell interaction with integrins-based cell-matrix interaction that controls motility, invasiveness and survival of cancer cells. Accordingly, here we examined silibinin effect on advanced human PCA PC3 cells' interaction with extracellular matrix component fibronectin. Silibinin (50-200 μM) treatment significantly decreased the fibronectin (5 μg/ml)-induced motile morphology via targeting actin cytoskeleton organization in PC3 cells. Silibinin also decreased the fibronectin-induced cell proliferation and motility but significantly increased cell death in PC3 cells. Silibinin also inhibited the PC3 cells invasiveness in Transwell invasion assays with fibronectin or cancer associated fibroblasts (CAFs) serving as chemoattractant. Importantly, PC3-luc cells cultured on fibronectin showed rapid dissemination and localized in lungs following tail vein injection in athymic male nude mice; however, in silibinin-treated PC3-luc cells, dissemination and lung localization was largely compromised. Molecular analyses revealed that silibinin treatment modulated the fibronectin-induced expression of integrins (α5, αV, β1 and β3), actin-remodeling (FAK, Src, GTPases, ARP2 and cortactin), apoptosis (cPARP and cleaved caspase 3), EMT (E-cadherin and β-catenin), and cell survival (survivin and Akt) related signaling molecules in PC3 cells. Furthermore, PC3-xenograft tissue analyses confirmed the inhibitory effect of silibinin on fibronectin and integrins expression. Together, these results showed that silibinin targets PCA cells' interaction with fibronectin and inhibits their motility, invasiveness and survival; thus further supporting silibinin use in PCA intervention including its metastatic progression.

Fibronectin regulates calvarial osteoblast differentiation

NASA Technical Reports Server (NTRS)

Moursi, A. M.; Damsky, C. H.; Lull, J.; Zimmerman, D.; Doty, S. B.; Aota, S.; Globus, R. K.

1996-01-01

The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM

PubMed Central

Hymes, Jeffrey P.; Johnson, Brant R.; Barrangou, Rodolphe

2016-01-01

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion of fbpB lost the ability to adhere to mucin and fibronectin in vitro. Homologues of fbpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside the L. acidophilus homology group. PMID:26921419

Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis

PubMed Central

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

2016-01-01

The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis. PMID:26811931

Recognition of fibronectin by Penicillium marneffei conidia via a sialic acid-dependent process and its relationship to the interaction between conidia and laminin.

PubMed

Hamilton, A J; Jeavons, L; Youngchim, S; Vanittanakom, N

1999-10-01

Adhesion of Penicillium marneffei conidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of P. marneffei conidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However, N-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of P. marneffei, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.

Plasma fibronectin: three steps to purification and stability.

PubMed

Poulouin, L; Gallet, O; Rouahi, M; Imhoff, J M

1999-10-01

Large amounts of soluble fibronectin were easily purified from cryoprecipitated or fresh citrated human blood plasma by a three-step combination of gelatin and heparin-cellufine affinity chromatography. The elution conditions were optimized to obtain a homogeneous fraction on SDS-PAGE and Western blot under reducing condition. No proteolytic activities were detected by zymography at acidic or neutral pH. Furthermore, the fibronectin preparation was stable over time up to 456 h at 37 degrees C in the presence of calcium, zinc, or mercury. This preparation of very stable fibronectin, called highly purified fibronectin (hpFN), gave a yield of 7.00 +/- 0.77 mg of fibronectin per gram of cryoprecipitated plasma and 0.16 mg of fibronectin per milliliter of fresh citrated, giving a yield of 32 to 53% (from presumed fibronectin concentration). This preparation may be useful for cellular tests and interaction analysis. Copyright 1999 Academic Press.

Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

NASA Technical Reports Server (NTRS)

Hughes-Fulford, M.; Gilbertson, V.

1999-01-01

The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

PubMed

Hocking, D C; Smith, R K; McKeown-Longo, P J

1996-04-01

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM.

PubMed

Hymes, Jeffrey P; Johnson, Brant R; Barrangou, Rodolphe; Klaenhammer, Todd R

2016-05-01

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion off bpB lost the ability to adhere to mucin and fibronectin in vitro Homologues off bpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside theL. acidophilus homology group. Copyright © 2016 Hymes et al.

Cellular origin of fibronectin in interspecies hybrid kidneys

PubMed Central

1984-01-01

The cellular origin of fibronectin in the kidney was studied in three experimental models. Immunohistochemical techniques that use cross- reacting or species-specific antibodies against mouse or chicken fibronectin were employed. In the first model studied, initially avascular mouse kidneys cultured on avian chorioallantoic membranes differentiate into epithelial kidney tubules and become vascularized by chorioallantoic vessels. Subsequently, hybrid glomeruli composed of mouse podocytes and avian endothelial-mesangial cells form. In immunohistochemical studies, cross-reacting antibodies to fibronectin stained vascular walls, tubular basement membranes, interstitium, and glomeruli of mouse kidney grafts. The species-specific antibodies reacting only with mouse fibronectin stained interstitial areas and tubular basement membranes, but showed no reaction with hybrid glomeruli and avian vascular walls. In contrast, species-specific antibodies against chicken fibronectin stained both the interstitial areas and the vascular walls as well as the endothelial-mesangial areas of the hybrid glomeruli, but did not stain the mouse-derived epithelial structures of the kidneys. In the second model, embryonic kidneys cultured under avascular conditions in vitro develop glomerular tufts, which are devoid of endothelial cells. These explants showed fluorescence staining for fibronectin only in tubular basement membranes and in interstitium. The avascular, purely epithelial glomerular bodies remained unstained. Finally, in outgrowths of separated embryonic glomeruli, the cross-reacting fibronectin antibodies revealed two populations of cells: one devoid of fibronectin and another expressing fibronectin in strong fibrillar and granular patterns. These results favor the idea that the main endogenous cellular sources for fibronectin in the embryonic kidney are the interstitial and vascular cells. All experiments presented here suggest that fibronectin is not synthesized by glomerular epithelial cells in vivo. PMID:6389571

The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression.

PubMed

Christopher, R A; Judge, S R; Vincent, P A; Higgins, P J; McKeown-Longo, P J

1999-10-01

Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin's amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by >50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin's amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.

Decorin inhibits cell migration through a process requiring its glycosaminoglycan side chain.

PubMed

Merle, B; Durussel, L; Delmas, P D; Clézardin, P

1999-12-01

Several studies overwhelmingly support the notion that decorin (DCN) is involved in matrix assembly, and in the control of cell adhesion and proliferation. However, nothing is known about the role of DCN during cell migration. Cell migration is a tightly regulated process which requires both adhesion (at the leading edge of the cell) and de-adhesion (at the trailing edge of the cell) from the substratum. We have determined in this study the effect of DCN on MG-63 osteosarcoma cell migration and have analyzed whether its effect is mediated by the protein core and/or the glycosaminoglycan side chain. DCN impeded the migration-promoting effect of matrix molecules (fibronectin, collagen type I) known to interact with the proteoglycan. Conversely, DCN did not counteract the migration-promoting effect of fibrinogen lacking proteoglycan affinity. DCN bearing dermatan-sulfate chains (i.e., skin and cartilage DCN) was about 20-fold more effective in inhibiting cell migration than DCN bearing chondroitin-sulfate chains (i.e., bone DCN). In addition, chondroitinase AC-treatment of cartilage DCN (which specifically removes chondroitin-sulfate chains) did not attenuate the inhibitory effect of this proteoglycan, while cartilage DCN deprived of both chondroitin- and dermatan-sulfate chains failed to alter cell migration promoted by either fibronectin or its heparin- and cell-binding domains. These data assert that the dermatan-sulfate chains of DCN are responsible for a negative influence on cell migration. However, isolated glycosaminoglycans failed to alter cell migration promoted by fibronectin, indicating that strongly negatively charged glycosaminoglycans alone cannot account for the impaired cell motility seen with DCN. Overall, these results show that the inhibitory action of DCN is dependent of substratum binding, is differentially mediated by its glycosaminoglycan side chains (chondroitin-sulfate vs. dermatan-sulfate chains), and is independent of a steric hindrance effect exerted by its glycosaminoglycan side chains. Copyright 1999 Wiley-Liss, Inc.

Sorption of fibronectin to human root surfaces in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mendieta, C.; Caravana, C.; Fine, D.H.

1990-05-01

The purpose of this study was to determine the conditions that favor the sorption and retention of human plasma fibronectin to cementum. Rectangular root segments prepared from teeth extracted for orthodontic reasons were mounted on a capillary pipette and immersed in solutions of {sup 125}I fibronectin for assay of cementum sorption under various conditions. Kinetic studies showed sorption to be rapid, with 77% of the maximum fibronectin sorption occurring within 1 minute. Fibronectin sorption was reduced when added in conjunction with serum and was inhibited by monovalent ions (such as sodium), but enhanced in the presence of divalent cations (suchmore » as calcium). Exposure of cementum to serum partially blocked subsequent sorption of fibronectin, while cementum bound fibronectin was eluted by subsequent exposure to serum. Treatment of cementum with citric acid pH 1.1 (4 minutes) followed by 5% sodium hypochlorite (5 minutes) caused a significant increase in fibronectin sorption with maximum retention upon subsequent exposure to serum (P less than 0.05). Fibronectin sorption to cementum was: rapid, electrostatic in nature, competitive, reversible, Ca+(+)-facilitated, and maximized by prior treatment of the root with citric acid and sodium hypochlorite. It is concluded that sorption of fibronectin to cementum can be achieved for clinical gain; however, conditions of application can significantly influence both accumulation and subsequent release of root sorbed material.« less

The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells.

PubMed

Molinari, G; Rohde, M; Talay, S R; Chhatwal, G S; Beckert, S; Podbielski, A

2001-04-01

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin-binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp-2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra-mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra-mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra-mutant bacteria. Adherence and internalization rates of a nra/speB-double mutant at wild-type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra-dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.

Culturing INS-1 cells on CDPGYIGSR-, RGD- and fibronectin surfaces improves insulin secretion and cell proliferation.

PubMed

Kuehn, Carina; Dubiel, Evan A; Sabra, Georges; Vermette, Patrick

2012-02-01

Rat insulinoma cells (INS-1), an immortalized pancreatic beta cell line, were cultured on low-fouling carboxymethyl-dextran (CMD) layers bearing fibronectin, the tripeptide Arg-Gly-Asp (RGD) or CDPGYIGSR, a laminin nonapeptide. INS-1 cells were non-adherent on CMD and RGE but adhered to fibronectin- and peptide-coated CMD surfaces and to tissue culture polystyrene (TCPS). On CMD bearing fibronectin and the peptides, INS-1 cells showed higher glucose-stimulated insulin secretion compared to those on TCPS, bare CMD and RGE. INS-1 cells experienced a net cell growth, with the lowest found after 7 days on CMD and the highest on fibronectin. Similarly, cells on RGD and CDPGYIGSR showed lower net growth rates than those on fibronectin. Expression of E-cadherin and integrins αvβ3 and α5 were similar between the conditions, except for α5 expression on fibronectin, RGD and CDPGYIGSR. Larger numbers of Ki-67-positive cells were found on CDPGYIGSR, TCPS, fibronectin and RGD. Cells in all conditions expressed Pdx1. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A fibronectin receptor on Candida albicans mediates adherence of the fungus to extracellular matrix

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klotz, S.A.; Smith, R.L.

1991-03-01

Binding of fibronectin, an extracellular matrix (ECM) protein, to Candida albicans was measured, and adherence of the fungus to immobilized ECM proteins, fibronectin, laminin, types I and IV collagen, and subendothelial ECM was studied. 125I-labeled fibronectin was inhibited from binding to the fungus by unlabeled human plasma fibronectin and by Arg-Gly-Asp (RGD), Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), and Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP), but binding was not inhibited by Gly-Arg-Gly-Asp-Ser-Pro. Soluble fibronectin, RGD, GRGESP, and GRGDTP also inhibited fungal adherence to the individual immobilized ECM proteins in a complex pattern, but only soluble fibronectin (10(-7) M) inhibited fungal adherence to subendothelial ECM. Thus, C. albicans possessesmore » at least one type of cell surface receptor for binding soluble fibronectin that can be inhibited with peptides. This receptor apparently is used to bind the fungus to immobilized ECM proteins and to subendothelial ECM and may play a role in the initiation of disseminated disease by bloodborne fungi by providing for adherence of the microorganisms to ECM proteins.« less

Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes.

PubMed

Hanski, E; Caparon, M

1992-07-01

Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.

Synergistic Effects of a Calcium Phosphate/Fibronectin Coating on the Adhesion of Periodontal Ligament Stem Cells Onto Decellularized Dental Root Surfaces.

PubMed

Lee, Jung-Seok; Kim, Hyun-Suk; Park, So-Yon; Kim, Tae-Wan; Jung, Jae-Suk; Lee, Jong-Bin; Kim, Chang-Sung

2015-01-01

This study aimed to enhance the attachment of periodontal ligament stem cells (PDLSCs) onto the decellularized dental root surface using surface coating with fibronectin and/or calcium phosphate (CaP) and to evaluate the activity of PDLSCs attached to a coated dental root surface following tooth replantation. PDLSCs were isolated from five dogs, and the other dental roots were used as a scaffold for carrying PDLSCs and then assigned to one of four groups according to whether their surface was coated with CaP, fibronectin, CaP/fibronectin, or left uncoated (control). Fibronectin increased the adhesion of PDLSCs onto dental root surfaces compared to both the control and CaP-coated groups, and simultaneous surface coating with CaP and fibronectin significantly accelerated and increased PDLSC adhesion compared to the fibronectin-only group. On in vivo tooth replantation, functionally oriented periodontal new attachment was observed on the CaP/fibronectin-coated dental roots to which autologous PDLSCs had adhered, while in the control condition, dental root replantation was associated only with root resorption and ankylosis along the entire root length. CaP and fibronectin synergistically enhanced the attachment of PDLSCs onto dental root surfaces, and autologous PDLSCs could produce de novo periodontal new attachment in an experimental in vivo model.

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

PubMed Central

Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

2012-01-01

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. PMID:22710062

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

PubMed

Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

2012-09-10

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

A new method to determine wound age in early vital skin injuries: a probability scoring system using expression levels of Fibronectin, CD62p and Factor VIII in wound hemorrhage.

PubMed

van de Goot, Franklin R W; Korkmaz, H Ibrahim; Fronczek, Judith; Witte, Birgit I; Visser, Rob; Ulrich, Magda M W; Begieneman, Mark P V; Rozendaal, Lawrence; Krijnen, Paul A J; Niessen, Hans W M

2014-11-01

In forensic autopsies it is important to determine the age of early vital skin wounds as accurate as possible. In addition to inflammation, coagulation is also induced in vital wounds. Analysis of blood coagulation markers in wound hemorrhage could therefore be an important additional discriminating factor in wound age determination. The aim of this study was to develop a wound age probability scoring system, based on the immunohistochemical expression levels of Fibronectin, CD62p and Factor VIII in wound hemorrhage. Tissue samples of (A) non injured control skin (n=383), and samples of mechanically induced skin injuries of known wound age, (B) injuries inflicted shortly before death (up to a few minutes old) (n=382), and (C) injuries inflicted 15-30 min before death (n=42) were obtained at autopsy in order to validate wound age estimation. Tissue slides were stained for Fibronectin, CD62p and Factor VIII and were subsequently scored for staining intensity (IHC score) in wound hemorrhage (1=minor, 2=moderate, 3=strong positive). Finally, probability scores of these markers were calculated. In at most 14% of the non-injured control samples, hemorrhage was found, with mean±standard deviation IHC scores of 0.1±0.4, 0.2±0.4 and 0.2±0.5 for Fibronectin, CD62p, and Factor VIII, respectively. Expression of these markers significantly increased to mean IHC scores 1.4±0.8 (Fibronectin), 1.2±0.6 (CD62p), and 1.6±0.8 (Factor VIII) in wounds inflicted shortly before death (few minutes old) and to 2.6±0.5 (Fibronectin), 2.5±0.6 (CD62p), and 2.8±0.4 (Factor VIII) in 15-30 min old wounds. The probabilities that a wound was non vital in case of an IH score 0 were 87%, 88% and 90% for Fibronectin, CD62p, and Factor VIII, respectively. In case of an IHC score 1 or 2, the probabilities that a wound was a few minutes old were 82/90%, 82/83% and 72/93%. Finally, in case of an IHC score 3, the probabilities that a wound was 15-30 min old were 65%, 76% and 55%. Based on the expression of Fibronectin, CD62p and Factor VIII in wound hemorrhage, we developed a probability scoring system that can be used in forensic autopsies to improve wound age estimation in early skin injuries. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The integrin expression profile modulates orientation and dynamics of force transmission at cell-matrix adhesions.

PubMed

Balcioglu, Hayri E; van Hoorn, Hedde; Donato, Dominique M; Schmidt, Thomas; Danen, Erik H J

2015-04-01

Integrin adhesion receptors connect the extracellular matrix (ECM) to the cytoskeleton and serve as bidirectional mechanotransducers. During development, angiogenesis, wound healing and cancer progression, the relative abundance of fibronectin receptors, including integrins α5β1 and αvβ3, changes, thus altering the integrin composition of cell-matrix adhesions. Here, we show that enhanced αvβ3 expression can fully compensate for loss of α5β1 and other β1 integrins to support outside-in and inside-out force transmission. α5β1 and αvβ3 each mediate actin cytoskeletal remodeling in response to stiffening or cyclic stretching of the ECM. Likewise, α5β1 and αvβ3 support cellular traction forces of comparable magnitudes and similarly increase these forces in response to ECM stiffening. However, cells using αvβ3 respond to lower stiffness ranges, reorganize their actin cytoskeleton more substantially in response to stretch, and show more randomly oriented traction forces. Centripetal traction force orientation requires long stress fibers that are formed through the action of Rho kinase (ROCK) and myosin II, and that are supported by α5β1. Thus, altering the relative abundance of fibronectin-binding integrins in cell-matrix adhesions affects the spatiotemporal organization of force transmission. © 2015. Published by The Company of Biologists Ltd.

Lectins as probes for assessing the accessibility of N-linked glycans in relation to the conformational changes of fibronectin.

PubMed

Agniel, Rémy; Vendrely, Charlotte; Poulouin, Laurent; Bascetin, Rümeyza; Benachour, Hamanou; Gallet, Olivier; Leroy-Dudal, Johanne

2015-12-01

Fibronectin, a ≈ 450-kDa protein with 4-9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal-induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose-binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal-induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research. Copyright © 2015 John Wiley & Sons, Ltd.

Propolis Modulates Fibronectin Expression in the Matrix of Thermal Injury

PubMed Central

Komosinska-Vassev, Katarzyna; Wisowski, Grzegorz; Mencner, Lukasz; Stojko, Jerzy; Kozma, Ewa M.

2014-01-01

The aim of the study was to assess the propolis effect on fibronectin metabolism in the course of burn wounds healing process. A model of burn wound healing of pig skin was applied. The amount of the released glycoprotein was assessed by a surface plasmon resonance. The profile of extracted fibronectin components was also assessed by an electrophoresis in polyacrylamide gel, with a subsequent immunodetection by Western Blotting. Propolis burn treatment decreased the release of fibronectin components from healing wounds in relation to damages treated with silver sulfadiazine. The main reason of decreased extraction of fibronectin components from wounds treated with propolis was a substantial decrease of degradation product release of the mentioned glycoprotein, which was observed particularly from the 3rd to 5th day of the repair. Wounds treatment with propolis demonstrated, especially in relation to damages treated with silver sulfadiazine, the decreased release of synthesized fibronectin molecules. The obtained results suggest that propolis modifies fibronectin metabolism in the course of wound healing process. The influence of propolis is reflected in prevention of fibronectin biosynthesis as well as its degradation in the wound area. The above-mentioned metabolic changes may decrease the risk of complications in the repair wounds process. PMID:24738072

Fibronectin in cell adhesion and migration via N-glycosylation

PubMed Central

Hsiao, Cheng-Te; Cheng, Hung-Wei; Huang, Chi-Ming; Li, Hao-Ru; Ou, Meng-Hsin; Huang, Jie-Rong; Khoo, Kay-Hooi; Yu, Helen Wenshin; Chen, Yin-Quan; Wang, Yang-Kao; Chiou, Arthur; Kuo, Jean-Cheng

2017-01-01

Directed cell migration is an important step in effective wound healing and requires the dynamic control of the formation of cell-extracellular matrix interactions. Plasma fibronectin is an extracellular matrix glycoprotein present in blood plasma that plays crucial roles in modulating cellular adhesion and migration and thereby helping to mediate all steps of wound healing. In order to seek safe sources of plasma fibronectin for its practical use in wound dressing, we isolated fibronectin from human (homo) and porcine plasma and demonstrated that both have a similar ability as a suitable substrate for the stimulation of cell adhesion and for directing cell migration. In addition, we also defined the N-glycosylation sites and N-glycans present on homo and porcine plasma fibronectin. These N-glycosylation modifications of the plasma fibronectin synergistically support the integrin-mediated signals to bring about mediating cellular adhesion and directed cell migration. This study not only determines the important function of N-glycans in both homo and porcine plasma fibronectin-mediated cell adhesion and directed cell migration, but also reveals the potential applications of porcine plasma fibronectin if it was applied as a material for clinical wound healing and tissue repair. PMID:29050309

Extensive vascular remodeling in the spinal cord of pre-symptomatic experimental autoimmune encephalomyelitis mice; increased vessel expression of fibronectin and the α5β1 integrin

PubMed Central

Boroujerdi, Amin; Welser-Alves, Jennifer V.; Milner, Richard

2013-01-01

Alterations in vascular structure and function are a central component of demyelinating disease. In addition to blood-brain barrier (BBB) breakdown, which occurs early in the course of disease, recent studies have described angiogenic remodeling, both in multiple sclerosis tissue and in the mouse demyelinating model, experimental autoimmune encephalomyelitis (EAE). As the precise timing of vascular remodeling in demyelinating disease has yet to be fully defined, the purpose of the current study was to define the time-course of these events in the MOG35-55 EAE model. Quantification of endothelial cell proliferation and vessel density revealed that a large part of angiogenic remodeling in cervical spinal cord white matter occurs during the pre-symptomatic phase of EAE. At the height of vascular remodeling, blood vessels in the cervical spinal cord showed strong transient upregulation of fibronectin and the α5β1 integrin. In vitro experiments revealed that α5 integrin inhibition reduced brain endothelial cell proliferation under inflammatory conditions. Interestingly, loss of vascular integrity was evident in all vessels during the first 4–7 days post-immunization, but after 14 days, was localized predominantly to venules. Taken together, our data demonstrate that extensive vascular remodeling occurs during the pre-symptomatic phase of EAE and point to a potential role for the fibronectin-α5β1 integrin interaction in promoting vascular remodeling during demyelinating disease. PMID:24056042

Extensive vascular remodeling in the spinal cord of pre-symptomatic experimental autoimmune encephalomyelitis mice; increased vessel expression of fibronectin and the α5β1 integrin.

PubMed

Boroujerdi, Amin; Welser-Alves, Jennifer V; Milner, Richard

2013-12-01

Alterations in vascular structure and function are a central component of demyelinating disease. In addition to blood-brain barrier (BBB) breakdown, which occurs early in the course of disease, recent studies have described angiogenic remodeling, both in multiple sclerosis tissue and in the mouse demyelinating model, experimental autoimmune encephalomyelitis (EAE). As the precise timing of vascular remodeling in demyelinating disease has yet to be fully defined, the purpose of the current study was to define the time-course of these events in the MOG35-55 EAE model. Quantification of endothelial cell proliferation and vessel density revealed that a large part of angiogenic remodeling in cervical spinal cord white matter occurs during the pre-symptomatic phase of EAE. At the height of vascular remodeling, blood vessels in the cervical spinal cord showed strong transient upregulation of fibronectin and the α5β1 integrin. In vitro experiments revealed that α5 integrin inhibition reduced brain endothelial cell proliferation under inflammatory conditions. Interestingly, loss of vascular integrity was evident in all vessels during the first 4-7days post-immunization, but after 14days, was localized predominantly to venules. Taken together, our data demonstrate that extensive vascular remodeling occurs during the pre-symptomatic phase of EAE and point to a potential role for the fibronectin-α5β1 integrin interaction in promoting vascular remodeling during demyelinating disease. © 2013.

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

PubMed

Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

2016-01-22

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions*

PubMed Central

Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A.; Brown, Elizabeth E.; Sanderson, Ralph D.

2016-01-01

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression. PMID:26601950

Inhibition of Pseudomonas aeruginosa adhesion to fibronectin by PA-IL and monosaccharides: involvement of a lectin-like process.

PubMed

Rebiere-Huët, Julie; Di Martino, Patrick; Hulen, Christian

2004-05-01

Pseudomonas aeruginosa adherence to fibronectin has been shown to be important to bacterial colonization and infection. To better understand the mechanisms involved in this interaction, the role of the carbohydrate moiety of the fibronectin molecule in P. aeruginosa adhesion was studied. Strain NK 125 502 adhered to immobilized fibronectin with an adherence index of 4.8 x 10(5) CFU/ micro g. Periodic oxidation of fibronectin markedly reduced the adhesion of P. aeruginosa, while a neuraminidase treatment increased bacteria adhesion. N-Acetylgalactosamine, N-acetylglucosamine, sialic acid, and also lectin PA-IL worked as efficient inhibitors in adhesion assays: 59%, 70.7%, 100%, and 60% of inhibition, respectively. We have demonstrated here the involvement of a lectin-like process in the interaction of P. aeruginosa NK 125 502 with immobilized fibronectin.

[The role of fibronectin in adhesion of corynebacteria to vaginal epitheliocytes].

PubMed

Gladysheva, I V; Cherkasov, S V

2014-01-01

Determination of the role of fibronectin in adhesion of corynebacteria to vaginal epitheliocytes. Corynebacterium genus microorganisms and primary epitheliocytes isolated from the lower reproductive tract of women were used. Adhesive ability of corynebacteria was studied in polystyrene plates against fixed fibronectin and on the model of vaginal epitheliocytes. Changes in adhesion of corynebacteria to vaginal epitheliocytes was evaluated after treatment of the latter with fibronectin. All the studied strains had the adhesion ability to fibronectin and vaginal epitheliocytes. The same strains were attributed to groups of high, moderate or low adhesive using both plate method and method utilizing vaginal epitheliocytes model, that tells of their comparability. During the addition of fibronectin to epitheliocytes, an enhancement of adhesion of all the studied corynebacteria strains took place. Adhesion index in strains isolated from healthy women increased by an average of 46.6%, adhesion index by 10.5 bact. cells/epith. In strains isolated from women with micro-ecologic disruption, adhesion increase was by 15.3% and 4.9 bact. cells/epith., respectively. Fibronectin is a factor that determines adhesion of corynebacteria to vaginal epitheliocytes and thus is important for formation of associative symbiosis of reproductive tract of women. The data obtained open perspective of use of fibronectin with the aim of colonizing ability increase of probiotics.

Tuberin Inhibits Production of the Matrix Protein Fibronectin in Diabetes

PubMed Central

Yadav, Mukesh; Tizani, Shaza; Bhandari, Basant; Valente, Anthony J.

2012-01-01

Exposure of proximal tubular epithelial cells to high glucose contributes to the accumulation of tubulointerstitial and matrix proteins in diabetic nephropathy, but how this occurs is not well understood. We investigated the effect of the signaling molecule tuberin, which modulates the mammalian target of rapamycin pathway, on renal hypertrophy and fibronectin expression. We found that the kidney mass was significantly greater in partially tuberin-deficient (TSC2+/−) diabetic rats than wild-type diabetic rats. Furthermore, TSC2+/− rats exhibited significant increases in the basal levels of phospho-tuberin and fibronectin expression in the kidney cortex. Increased levels of phosphorylated tuberin associated with an increase in fibronectin expression in both wild-type and TSC2+/− diabetic rats. Treatment with insulin abrogated the diabetes-induced increase in fibronectin expression. In vitro, high glucose enhanced fibronectin expression in TSC2+/− primary proximal tubular epithelial cells; both inhibition of Akt and inhibition of the mammalian target of rapamycin could prevent this effect of glucose. In addition, forced expression of tuberin in tuberin-null cells abolished the expression of fibronectin protein. Taken together, these data suggest that tuberin plays a central role in the development of renal hypertrophy and in modulating the production of the matrix protein fibronectin in diabetes. PMID:22904348

MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization

PubMed Central

Hayakawa, Tohru; Yoshida, Eiji; Yoshimura, Yoshitaka; Uo, Motohiro; Yoshinari, Masao

2012-01-01

The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0 nm) and sandblasting (Ra: approximately 1.0 μm), and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells. PMID:22675359

Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

PubMed Central

Yamada, Aya; Yuasa, Kenji; Yoshizaki, Keigo; Iwamoto, Tsutomu; Saito, Masahiro; Nakamura, Takashi; Fukumoto, Satoshi

2015-01-01

The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation. PMID:25830530

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins.

PubMed

Rebière-Huët, J; Guérillon, J; Pimenta, A L; Di Martino, P; Orange, N; Hulen, C

2002-09-24

Bacterial adherence is a complex phenomenon involving specific interactions between receptors, including matricial fibronectin, and bacterial ligands. We show here that fibronectin and outer membrane proteins of Pseudomonas fluorescens were able to inhibit adherence of P. fluorescens to fibronectin-coated wells. We identified at least six fibronectin-binding proteins with molecular masses of 70, 55, 44, 37, 32 and 28 kDa. The presence of native (32 kDa) and heat-modified forms (37 kDa) of OprF was revealed by immuno-analysis and the 44-kDa band was composed of three proteins, their N-terminal sequences showing homologies with Pseudomonas aeruginosa porins (OprD, OprE1 and OprE3).

Corrosion resistance and biocompatibility of magnesium alloy modified by alkali heating treatment followed by the immobilization of poly (ethylene glycol), fibronectin and heparin.

PubMed

Pan, Changjiang; Hu, Youdong; Hou, Yu; Liu, Tao; Lin, Yuebin; Ye, Wei; Hou, Yanhua; Gong, Tao

2017-01-01

In recent years, magnesium alloys are attracting more and more attention as a kind of biodegradable metallic biomaterials, however, their uncontrollable biodegradation speed in vivo and the limited surface biocompatibility hinder their clinical applications. In the present study, with the aim of improving the corrosion resistance and biocompatibility, the magnesium alloy (AZ31B) surface was modified by alkali heating treatment followed by the self-assembly of 3-aminopropyltrimethoxysilane (APTMS). Subsequently, poly (ethylene glycol) (PEG) and fibronectin or fibronectin/heparin complex were sequentially immobilized on the modified surface. The results of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed that the above molecules were successfully immobilized on the magnesium alloy surface. An excellent hydrophilic surface was obtained after the alkali heating treatment while the hydrophilicity decreased to some degree after the self-assembly of APTMS, the surface hydrophilicity was gradually improved again after the immobilization of PEG, fibronectin or fibronectin/heparin complex. The corrosion resistance of the control magnesium alloy was significantly improved by the alkali heating treatment. The self-assembly of APTMS and the following immobilization of PEG further enhanced the corrosion resistance of the substrates, however, the grafting of fibronectin or fibronectin/heparin complex slightly lowered the corrosion resistance. As compared to the pristine magnesium alloy, the samples modified by the immobilization of PEG and fibronectin/heparin complex presented better blood compatibility according to the results of hemolysis assay and platelet adhesion as well as the activated partial thromboplastin time (APTT). In addition, the modified substrates had better cytocompatibility to endothelial cells due to the improved anticorrosion and the introduction of fibronectin. The substrates modified by fibronectin or fibronectin/heparin complex can significantly promote endothelial cell adhesion and proliferation. Taking all these results into consideration, the method of the present study can be used for the surface modification of the magnesium alloy to simultaneously impart it better corrosion resistance, favorable blood compatibility and good cytocompatibility to endothelial cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Disruption of fibronectin matrix affects type IV collagen, fibrillin and laminin deposition into extracellular matrix of human trabecular meshwork (HTM) cells.

PubMed

Filla, Mark S; Dimeo, Kaylee D; Tong, Tiegang; Peters, Donna M

2017-12-01

Fibronectin fibrils are a major component of the extracellular matrix (ECM) of the trabecular meshwork (TM). They are a key mediator of the formation of the ECM which controls aqueous humor outflow and contributes to the pathogenesis of glaucoma. The purpose of this work was to determine if a fibronectin-binding peptide called FUD, derived from the Streptococcus pyogenes Functional Upstream Domain of the F1 adhesin protein, could be used to control fibronectin fibrillogenesis and hence ECM formation under conditions where its expression was induced by treatment with the glucocorticoid dexamethasone. FUD was very effective at preventing fibronectin fibrillogenesis in the presence or absence of steroid treatment as well as the removal of existing fibronectin fibrils. Disruption of fibronectin fibrillogenesis by FUD also disrupted the incorporation of type IV collagen, laminin and fibrillin into the ECM. The effect of FUD on these other protein matrices, however, was found to be dependent upon the maturity of the ECM when FUD was added. FUD effectively disrupted the incorporation of these other proteins into matrices when added to newly confluent cells that were forming a nascent ECM. In contrast, FUD had no effect on these other protein matrices if the cell cultures already possessed a pre-formed, mature ECM. Our studies indicate that FUD can be used to control fibronectin fibrillogenesis and that these fibrils play a role in regulating the assembly of other ECM protein into matrices involving type IV collagen, laminin, and fibrillin within the TM. This suggests that under in vivo conditions, FUD would selectively disrupt fibronectin fibrils and de novo assembly of other proteins into the ECM. Finally, our studies suggest that targeting fibronectin fibril assembly may be a viable treatment for POAG as well as other glaucomas involving excessive or abnormal matrix deposition of the ECM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism

PubMed Central

Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M.; Sottile, Jane

2014-01-01

Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein fibronectin polymerization promotes the SMC synthetic phenotype by modulating the expression of smooth muscle cell differentiation proteins. PMID:24752318

Glucocorticoid-induced skeletal muscle atrophy in vitro is attenuated by mechanical stimulation

NASA Technical Reports Server (NTRS)

Chromiak, J. A.; Vandenburgh, H. H.

1992-01-01

Glucocorticoids induce rapid atrophy of fast skeletal myofibers in vivo, and either weight lifting or endurance exercise reduces this atrophy by unknown mechanisms. We examined the effects of the synthetic glucocorticoid dexamethasone (Dex) on protein turnover in tissue-cultured avian fast skeletal myofibers and determined whether repetitive mechanical stretch altered the myofiber response to Dex. In static cultures after 3-5 days, 10(-8) M Dex decreased total protein content 42-74%, total protein synthesis rates 38-56%, mean myofiber diameter 35%, myosin heavy chain (MHC) content 86%, MHC synthesis rate 44%, and fibronectin synthesis rate 29%. Repetitive 10% stretch-relaxations of the cultured myofibers for 60 s every 5 min for 3-4 days prevented 52% of the Dex-induced decrease in protein content, 42% of the decrease in total protein synthesis rate, 77% of the decrease in MHC content, 42% of the decrease in MHC synthesis rate, and 67% of the decrease in fibronectin synthesis rate. This in vitro model system will complement in vivo studies in understanding the mechanism by which mechanical activity and glucocorticoids interact to regulate skeletal muscle growth.

Binding of the fibronectin-mimetic peptide, PR_b, to α5β1 on pig islet cells increases fibronectin production and facilitates internalization of PR_b functionalized liposomes

PubMed Central

Atchison, Nicole A.; Fan, Wei; Papas, Klearchos K.; Hering, Bernhard J.; Tsapatsis, Michael; Kokkoli, Efrosini

2010-01-01

Islet transplantation is a promising treatment for type 1 diabetes. Recent studies have demonstrated that human islet allografts can restore insulin independence to patients with this disease. As islet isolation and immunotherapeutic techniques improve, the demand for this cell-based therapy will dictate the need for other sources of islets. Pig islets could provide an unlimited supply for xenotransplantation and have shown promise as an alternative to human islet allografts. However, stresses imposed during islet isolation and transplantation decrease islet viability, leading to loss of graft function. In this study, we investigated the ability of a fibronectin-mimetic peptide, PR_b, which specifically binds to the α5β1 integrin, to reestablish lost extracellular matrix (ECM) around isolated pig islets and increase internalization of liposomes. Confocal microscopy and western blotting were used to show the presence of the integrin α5β1 on the pig islets on day 0 (day of isolation), as well as different days of islet culture. Islets cultured in medium supplemented with free PR_b for 48 hours were found to have increased levels of ECM fibronectin secretion compared to islets in normal culture conditions. Using confocal microscopy and flow cytometry we found that PR_b peptide-amphiphile functionalized liposomes delivered to the pig islets internalized into the cells in a PR_b concentration dependent manner, and non-functionalized liposomes showed minimal internalization. These studies proved that the fibronectin-mimetic peptide, PR_b, is an appropriate peptide bullet for applications involving α5β1 expressing pig islet cells. Fibronectin production stimulated through α5β1 PR_b binding may decrease apoptosis and therefore increase islet viability in culture. In addition, PR_b peptide-amphiphile functionalized liposomes may be used for targeted delivery of different agents to pig islet cells. PMID:20704278

The glycation of fibronectin by glycolaldehyde and methylglyoxal as a model for aging in Bruch's membrane.

PubMed

Thao, Mai T; Gaillard, Elizabeth R

2016-07-01

The purpose of the study is to identify the sites of modification when fibronectin reacts with glycolaldehyde or methylglyoxal as a model system for aging of Bruch's membrane. A synthetic peptide consisting of the α5β1 integrin binding region of fibronectin was incubated with glycolaldehyde for 12 h or with methylglyoxal for 1 h at 37 °C. After tryptic digestion, the samples were analyzed with liquid chromatography-mass spectrometry (LC/MS). Tandem MS was used to determine the sites of modification. The adducts, aldoamine and N (ε)-carboxymethyl-lysine, attached preferably at lysine residues when the fibronectin peptide reacted with glycolaldehyde. When the fibronectin peptide reacted with methylglyoxal, modifications occurred at lysine and arginine residues. At lysine residues, N (ε)-carboxyethyl-lysine adducts were present. At arginine residues, hydroimidazolone and tetrapyrimidine adducts were present. Several advanced glycation endproducts were generated when fibronectin was glycated via glycolaldehyde and methylglyoxal. These results can help explain the structural changes Bruch's membrane undergoes during aging.

The Fibronectin-Binding Protein Fnm Contributes to Adherence to Extracellular Matrix Components and Virulence of Enterococcus faecium

PubMed Central

Somarajan, Sudha R.; La Rosa, Sabina Leanti; Singh, Kavindra V.; Roh, Jung H.; Höök, Magnus

2015-01-01

The interaction between bacteria and fibronectin is believed to play an important role in the pathogenicity of clinically important Gram-positive cocci. In the present study, we identified a gene encoding a predicted fibronectin-binding protein of Enterococcus faecium (fnm), a homologue of Streptococcus pneumoniae pavA, in the genomes of E. faecium strain TX82 and all other sequenced E. faecium isolates. Full-length recombinant Fnm from strain TX82 bound to immobilized fibronectin in a concentration-dependent manner and also appeared to bind collagen type V and laminin, but not other proteins, such as transferrin, heparin, bovine serum albumin, mucin, or collagen IV. We demonstrated that the N-terminal fragment of Fnm is required for full fibronectin binding, since truncation of this region caused a 2.4-fold decrease (P < 0.05) in the adhesion of E. faecium TX82 to fibronectin. Deletion of fnm resulted in a significant reduction (P < 0.001) in the ability of the mutant, TX6128, to bind fibronectin relative to that of the wild-type strain; in situ reconstitution of fnm in the deletion mutant strain restored adherence. In addition, the Δfnm mutant was highly attenuated relative to TX82 (P ≤ 0.0001) in a mixed-inoculum rat endocarditis model. Taken together, these results demonstrate that Fnm affects the adherence of E. faecium to fibronectin and is important in the pathogenesis of experimental endocarditis. PMID:26371130

Fibronectin on extracellular vesicles from microvascular endothelial cells is involved in the vesicle uptake into oligodendrocyte precursor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osawa, Sho; Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511; Kurachi, Masashi

We previously reported transplantation of brain microvascular endothelial cells (MVECs) into cerebral white matter infarction model improved the animal's behavioral outcome by increasing the number of oligodendrocyte precursor cells (OPCs). We also revealed extracellular vesicles (EVs) derived from MVECs promoted survival and proliferation of OPCs in vitro. In this study, we investigated the mechanism how EVs derived from MVECs contribute to OPC survival and proliferation. Protein mass spectrometry and enzyme-linked immunosorbent assay revealed fibronectin was abundant on the surface of EVs from MVECs. As fibronectin has been reported to promote OPC survival and proliferation via integrin signaling pathway, we blocked themore » binding between fibronectin and integrins using RGD sequence mimics. Blocking the binding, however, did not attenuate the survival and proliferation promoting effect of EVs on OPCs. Flow cytometric and imaging analyses revealed fibronectin on EVs mediates their internalization into OPCs by its binding to heparan sulfate proteoglycan on OPCs. OPC survival and proliferation promoted by EVs were attenuated by blocking the internalization of EVs into OPCs. These lines of evidence suggest that fibronectin on EVs mediates their internalization into OPCs, and the cargo of EVs promotes survival and proliferation of OPCs, independent of integrin signaling pathway. - Highlights: • Fibronectin exists on the surface of extracellular vesicles from endothelial cells. • Integrin signaling is not involved in effects of extracellular vesicles on OPCs. • Fibronectin on the surface of extracellular vesicles mediates their uptake into OPCs.« less

Development of a microfabricated artificial limbus with micropockets for cell delivery to the cornea.

PubMed

Ortega, Ílida; Deshpande, Pallavi; Gill, Andrew A; MacNeil, Sheila; Claeyssens, Frederik

2013-06-01

The aim of this study was to develop a synthetic alternative to the human corneal limbus for use initially as an ex vivo model in which to study corneal stem cell function within a niche environment and ultimately to develop an implantable limbus for future clinical use. Microstereolithography was used for the fabrication of polyethylene glycol diacrylate (PEGDA) based rings on a macroscopic (1.2 cm) scale containing unique microfeatures (pockets) which were then modified with fibronectin to promote cell adhesion. These rings were designed to mimic the limbal area of the eye containing structures of the approximate size and shape of the stem cell microenvironments found in the palisades of Vogt. The attachment of rabbit limbal fibroblasts and rabbit limbal epithelial cells to the PEGDA rings was increased by pretreating the microfabricated structures with biotinylated fibronectin. Cell outgrowth from fibronectin coated microfabricated structures was 50% greater than from rings without structures or fibronectin coating. The cell loaded rings were then placed on an ex vivo wounded cornea model and the outgrowth of cells to form a multilayered epithelium was observed. We suggest this is a new approach to investigating limbal stem cells niches and the first steps towards a new approach for corneal regeneration.

In Vitro Analysis of Fibronectin-Modified Titanium Surfaces

PubMed Central

Chang, Yu-Chi; Lee, Wei-Fang; Feng, Sheng-Wei; Huang, Haw-Ming; Lin, Che-Tong; Teng, Nai-Chia; Chang, Wei Jen

2016-01-01

Background Glow discharge plasma (GDP) procedure is an effective method for grafting various proteins, including albumin, type I collagen, and fibronectin, onto a titanium surface. However, the behavior and impact of titanium (Ti) surface modification is yet to be unraveled. Purpose The purpose of this study is to evaluate and analyze the biological properties of fibronectin-grafted Ti surfaces treated by GDP. Materials and Methods Grade II Ti discs were initially cleaned and autoclaved to obtain original specimens. Subsequently, the specimens were GDP treated and grafted with fibronectin to form Ar-GDP (Argon GDP treatment only) and GDP-fib (fibronectin coating following GDP treatment) groups. Blood coagulation test and MG-63 cell culture were performed to evaluate the biological effects on the specimen. Results There was no significant difference between Ar-GDP and GDP-fib groups in blood compatibility analysis. While in the MTT test, cellular proliferation was benefited from the presence of fibronectin coating. The numbers of cells on Ar-GDP and GDP-fib specimens were greater than those in the original specimens after 24 h of culturing. Conclusions GDP treatment combined with fibronectin grafting favored MG-63 cell adhesion, migration, and proliferation on titanium surfaces, which could be attributed to the improved surface properties. PMID:26731536

Fibronectin matrix assembly is essential for cell condensation during chondrogenesis

PubMed Central

Singh, Purva; Schwarzbauer, Jean E.

2014-01-01

ABSTRACT Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation. PMID:25146392

Fibronectin matrix assembly is essential for cell condensation during chondrogenesis.

PubMed

Singh, Purva; Schwarzbauer, Jean E

2014-10-15

Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation. © 2014. Published by The Company of Biologists Ltd.

Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin.

PubMed

Spahich, Nicole A; Kenjale, Roma; McCann, Jessica; Meng, Guoyu; Ohashi, Tomoo; Erickson, Harold P; St Geme, Joseph W

2014-06-01

Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1-2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS-fibronectin interaction. © 2014 The Authors.

Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin

PubMed Central

Spahich, Nicole A.; Kenjale, Roma; McCann, Jessica; Meng, Guoyu; Ohashi, Tomoo; Erickson, Harold P.

2014-01-01

Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1–2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS–fibronectin interaction. PMID:24687948

Women’s perspectives of the fetal fibronectin testing process: a qualitative descriptive study

PubMed Central

2014-01-01

Background In 2009 the Ontario Ministry of Health and Long Term Care funded the implementation of province-wide fetal fibronectin testing in Ontario hospitals. This paper reports results from the provincial evaluation that sought to describe the experience of fetal fibronectin testing from the perspective of women with symptoms of preterm labour. Methods A descriptive qualitative design was used, employing semi-structured telephone and face-to-face interviews with women who had fetal fibronectin testing. Results Five hospitals participated in recruiting women for the study and 17 women were interviewed. Women described their experiences of fetal fibronectin testing as an emotional process that moves from expecting, to feeling, to hoping for reassurance; and then to re-defining what is required to feel reassured. Women described feeling anxious while waiting for fetal fibronectin results. When test results were negative, women described feeling a sense of relief that their symptoms would not likely lead to an imminent preterm birth. Women with positive results expressed feeling reassured by the care decisions and quick action taken by the health care team. Conclusion Fetal fibronectin testing was acceptable and beneficial to these women with symptoms of preterm labour. Implications for practice and future research are suggested. PMID:24894630

The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

PubMed

Hashim, Puziah

2014-03-01

Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

NASA Technical Reports Server (NTRS)

Moursi, A. M.; Globus, R. K.; Damsky, C. H.

1997-01-01

We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

Role of a bacillus Calmette-Guérin fibronectin attachment protein in BCG-induced antitumor activity.

PubMed

Zhao, W; Schorey, J S; Bong-Mastek, M; Ritchey, J; Brown, E J; Ratliff, T L

2000-04-01

Intravesical Mycobacterium bovis bacillus Calmette-Gu*erin (BCG) is the treatment of choice for superficial bladder cancer. Previous studies showed that attachment of BCG to fibronectin within the bladder was necessary for mediation of the antitumor response. Further studies identified a bacterial receptor, fibronectin attachment protein (FAP), as an important mediator of BCG attachment to fibronectin. In vitro studies showed that a stable BCG/fibronectin interaction was dependent on FAP binding to fibronectin; however, no role for FAP in the attachment of BCG in vivo has been characterized. We now report the cloning of the M. bovis BCG FAP (FAP-B) and demonstrate an important role for FAP in the in vivo attachment of BCG to the bladder wall and in the induction of BCG-mediated antitumor activity. The predicted amino acid sequence for FAP-B shows 61% and 71% homology, respectively, with Mycobacterium avium FAP (FAP-A) and Mycobacterium leprae FAP (FAP-L). Rabbit polyclonal antibodies against Mycobacterium vaccae FAP (FAP-V) reacted with all 3 recombinant FAP proteins on Western blots. Functional studies show FAP-B to bind fibronectin via the highly conserved attachment regions previously identified for FAP-A and FAP-L and also to competitively inhibit attachment of BCG to matrix fibronectin. In vivo studies show FAP to be a necessary protein for the stable attachment of BCG to the bladder wall. Moreover, stable binding of BCG via FAP was shown to be necessary for the expression of BCG-induced antitumor activity. Our results demonstrate a biological role for FAP in the mediation of BCG-induced antitumor activity.

The transcription factor ETS-1 regulates angiotensin II-stimulated fibronectin production in mesangial cells.

PubMed

Hua, Ping; Feng, Wenguang; Rezonzew, Gabriel; Chumley, Phillip; Jaimes, Edgar A

2012-06-01

Angiotensin II (ANG II) produced as result of activation of the renin-angiotensin system (RAS) plays a critical role in the pathogenesis of chronic kidney disease via its hemodynamic effects on the renal microcirculation as well as by its nonhemodynamic actions including the production of extracellular matrix proteins such as fibronectin, a multifunctional extracellular matrix protein that plays a major role in cell adhesion and migration as well as in the development of glomerulosclerosis. ETS-1 is an important transcription factor essential for normal kidney development and glomerular integrity. We previously showed that ANG II increases ETS-1 expression and is required for fibronectin production in mesangial cells. In these studies, we determined that ANG II induces phosphorylation of ETS-1 via activation of the type 1 ANG II receptor and that Erk1/2 and Akt/PKB phosphorylation are required for these effects. In addition, we characterized the role of ETS-1 on the transcriptional activation of fibronectin production in mesangial cells. We determined that ETS-1 directly activates the fibronectin promoter and by utilizing gel shift assays and chromatin immunoprecipitation assays identified two different ETS-1 binding sites that promote the transcriptional activation of fibronectin in response to ANG II. In addition, we identified the essential role of CREB and its coactivator p300 on the transcriptional activation of fibronectin by ETS-1. These studies unveil novel mechanisms involved in RAS-induced production of the extracellular matrix protein fibronectin in mesangial cells and establish the role of the transcription factor ETS-1 as a direct mediator of these effects.

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

PubMed

Llewellyn-Jones, C G; Lomas, D A; Stockley, R A

1994-06-01

Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage.

Fibronectin non-amyloid glomerulopathy.

PubMed

Yong, Jim L; Killingsworth, Murray C; Spicer, S Timothy; Wu, Xiao-Juan

2009-11-20

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 micromol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology.

Fibronectin non-amyloid glomerulopathy

PubMed Central

Yong, Jim L; Killingsworth, Murray C; Spicer, S Timothy; Wu, Xiao-Juan

2010-01-01

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 µmol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology. PMID:20126589

Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling

PubMed Central

Yun, Sanguk; Budatha, Madhusudhan; Dahlman, James E.; Coon, Brian G.; Cameron, Ryan T.; Langer, Robert; Anderson, Daniel G.; Baillie, George; Schwartz, Martin A.

2016-01-01

Atherosclerosis is primarily a disease of lipid metabolism and inflammation; however, it is also closely associated with endothelial extracellular matrix (ECM) remodelling, with fibronectin accumulating in the laminin–collagen basement membrane. To investigate how fibronectin modulates inflammation in arteries, we replaced the cytoplasmic tail of the fibronectin receptor integrin α5 with that of the collagen/laminin receptor integrin α2. This chimaera suppressed inflammatory signalling in endothelial cells on fibronectin and in knock-in mice. Fibronectin promoted inflammation by suppressing anti-inflammatory cAMP. cAMP was activated through endothelial prostacyclin secretion; however, this was ECM-independent. Instead, cells on fibronectin suppressed cAMP via enhanced phosphodiesterase (PDE) activity, through direct binding of integrin α5 to phosphodiesterase-4D5 (PDE4D5), which induced PP2A-dependent dephosphorylation of PDE4D5 on the inhibitory site Ser651. In vivo knockdown of PDE4D5 inhibited inflammation at athero-prone sites. These data elucidate a molecular mechanism linking ECM remodelling and inflammation, thereby identifying a new class of therapeutic targets. PMID:27595237

Crystal structure of tandem type III fibronectin domains from Drosophila neuroglian at 2.0 A.

PubMed

Huber, A H; Wang, Y M; Bieber, A J; Bjorkman, P J

1994-04-01

We report the crystal structure of two adjacent fibronectin type III repeats from the Drosophila neural cell adhesion molecule neuroglian. Each domain consists of two antiparallel beta sheets and is folded topologically identically to single fibronectin type III domains from the extracellular matrix proteins tenascin and fibronectin. beta bulges and left-handed polyproline II helices disrupt the regular beta sheet structure of both neuroglian domains. The hydrophobic interdomain interface includes a metal-binding site, presumably involved in stabilizing the relative orientation between domains and predicted by sequence comparision to be present in the vertebrate homolog molecule L1. The neuroglian domains are related by a near perfect 2-fold screw axis along the longest molecular dimension. Using this relationship, a model for arrays of tandem fibronectin type III repeats in neuroglian and other molecules is proposed.

Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice.

PubMed

Kitasaka, Hiroya; Kawai, Tomoko; Hoque, S A Masudul; Umehara, Takashi; Fujita, Youko; Shimada, Masayuki

2018-01-01

It has been known that EGF-like factor secreted from LH-stimulated granuloma cells acts on granulosa cells and cumulus cells to induce ovulation process. Granulosa cells are changed the morphology with differentiating cell functions to produce progesterone. Cumulus cells are detached to make a space between the cells to accumulate hyaluronan rich matrix. LH also changes extracellular matrix (ECM) components including fibronectin in the follicular walls and granulosa cell layers. EGF like factor and fibronectin synergistically play important roles in numerous cell functions, especially cancer cell migration, estimating that fibronectin would impact on granulosa cells and cumulus cells. To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. The functions were monitored by the detection of downstream signaling pathway, focal adhesion kinase (FAK). The pharmacological approach in both in vivo and in vitro were used for analyzing the physiological roles of FAK during ovulation process. The immunofluorescence staining revealed that fibronectin and integrin were observed in granulosa cells, cumulus cells and the space between cumulus cells and oocyte at 4 and 8 h after hCG injection. Concomitantly with the changes of fibronectin-integrin localization, FAK was phosphorylated in periovulatory follicles. The injection of FAK inhibitor suppressed not only ovulation but also luteinization of granulosa cells and cumulus expansion. In cultured-granulosa cells, fibronectin-integrin synergistically activated FAK with amphiregulin (AREG). Such cooperative stimulations induced a morphological change in granulosa cells, which resulted in the maximum level of progesterone production via the induction of Hsd3b. When cumulus-oocyte complexes (COCs) were cultured with AREG in the presence of serum, the maximum level of cumulus expansion was observed. The AREG-induced cumulus expansion was also suppressed by FAK inhibitor. Thus, it is concluded that fibronectin and AREG synergistically activate FAK not only in granulosa cells and cumulus cells to induce successful ovulation process.

Fibronectin-based scaffold domain proteins that bind myostatin: a patent evaluation of WO2014043344.

PubMed

Walker, Ryan G; Thompson, Thomas B

2015-05-01

Muscular dystrophies (MD) are commonly characterized by progressive loss of muscle mass and function. It is hypothesized that therapeutic blockade of the TGF-β ligand myostatin, a negative regulator of muscle mass, will stimulate muscle growth and restore muscle function. Although many anti-myostatin targets are currently being pursued in the clinical setting, the efficacies of the tested molecules have shown mixed results. The patent WO2014043344 describes a novel approach for myostatin inhibition using a modified fibronectin type III domain that could potentially be used to treat MD and other muscle-related pathologies.

Living biointerfaces based on non-pathogenic bacteria support stem cell differentiation

NASA Astrophysics Data System (ADS)

Hay, Jake J.; Rodrigo-Navarro, Aleixandre; Hassi, Karoliina; Moulisova, Vladimira; Dalby, Matthew J.; Salmeron-Sanchez, Manuel

2016-02-01

Lactococcus lactis, a non-pathogenic bacteria, has been genetically engineered to express the III7-10 fragment of human fibronectin as a membrane protein. The engineered L. lactis is able to develop biofilms on different surfaces (such as glass and synthetic polymers) and serves as a long-term substrate for mammalian cell culture, specifically human mesenchymal stem cells (hMSC). This system constitutes a living interface between biomaterials and stem cells. The engineered biofilms remain stable and viable for up to 28 days while the expressed fibronectin fragment induces hMSC adhesion. We have optimised conditions to allow long-term mammalian cell culture, and found that the biofilm is functionally equivalent to a fibronectin-coated surface in terms of osteoblastic differentiation using bone morphogenetic protein 2 (BMP-2) added to the medium. This living bacteria interface holds promise as a dynamic substrate for stem cell differentiation that can be further engineered to express other biochemical cues to control hMSC differentiation.

A biosensor generated via high throughput screening quantifies cell edge Src dynamics

PubMed Central

Gulyani, Akash; Vitriol, Eric; Allen, Richard; Wu, Jianrong; Gremyachinskiy, Dmitriy; Lewis, Steven; Dewar, Brian; Graves, Lee M.; Kay, Brian K.; Kuhlman, Brian; Elston, Tim; Hahn, Klaus M.

2011-01-01

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge. PMID:21666688

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

PubMed Central

Llewellyn-Jones, C. G.; Lomas, D. A.; Stockley, R. A.

1994-01-01

BACKGROUND--Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. METHODS--The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. RESULTS--When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. CONCLUSIONS--It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage. Images PMID:7912452

Fibronectins containing extradomain A or B enhance osteoblast differentiation via distinct integrins

PubMed Central

Sens, Carla; Huck, Katrin; Pettera, Stefan; Uebel, Stephan; Wabnitz, Guido; Moser, Markus; Nakchbandi, Inaam A.

2017-01-01

Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4β1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to β3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation. PMID:28325836

BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells

PubMed Central

Klamer, Sofieke E; Kuijk, Carlijn GM; Hordijk, Peter L; van der Schoot, C Ellen; von Lindern, Marieke; van Hennik, Paula B; Voermans, Carlijn

2013-01-01

Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis. PMID:24152593

Proteomic Analysis of Gingival Tissue and Alveolar Bone during Alveolar Bone Healing*

PubMed Central

Yang, Hee-Young; Kwon, Joseph; Kook, Min-Suk; Kang, Seong Soo; Kim, Se Eun; Sohn, Sungoh; Jung, Seunggon; Kwon, Sang-Oh; Kim, Hyung-Seok; Lee, Jae Hyuk; Lee, Tae-Hoon

2013-01-01

Bone tissue regeneration is orchestrated by the surrounding supporting tissues and involves the build-up of osteogenic cells, which orchestrate remodeling/healing through the expression of numerous mediators and signaling molecules. Periodontal regeneration models have proven useful for studying the interaction and communication between alveolar bone and supporting soft tissue. We applied a quantitative proteomic approach to analyze and compare proteins with altered expression in gingival soft tissue and alveolar bone following tooth extraction. For target identification and validation, hard and soft tissue were extracted from mini-pigs at the indicated times after tooth extraction. From triplicate experiments, 56 proteins in soft tissue and 27 proteins in alveolar bone were found to be differentially expressed before and after tooth extraction. The expression of 21 of those proteins was altered in both soft tissue and bone. Comparison of the activated networks in soft tissue and alveolar bone highlighted their distinct responsibilities in bone and tissue healing. Moreover, we found that there is crosstalk between identified proteins in soft tissue and alveolar bone with respect to cellular assembly, organization, and communication. Among these proteins, we examined in detail the expression patterns and associated networks of ATP5B and fibronectin 1. ATP5B is involved in nucleic acid metabolism, small molecule biochemistry, and neurological disease, and fibronectin 1 is involved in cellular assembly, organization, and maintenance. Collectively, our findings indicate that bone regeneration is accompanied by a profound interaction among networks regulating cellular resources, and they provide novel insight into the molecular mechanisms involved in the healing of periodontal tissue after tooth extraction. PMID:23824910

Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites.

PubMed

Klotzsch, Enrico; Smith, Michael L; Kubow, Kristopher E; Muntwyler, Simon; Little, William C; Beyeler, Felix; Gourdon, Delphine; Nelson, Bradley J; Vogel, Viola

2009-10-27

Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stress-strain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed.

Fibronectin gene polymorphism in patients with lung cancer.

PubMed

Siemianowicz, K; Gminski, J; Francuz, T; Syzdol, M; Polanska, D; Machalski, M; Brulinski, K; Magiera-Molendowska, H

2001-01-01

Fibronectin (FN) is a glycoprotein component of connective tissue. It is involved in cancer progression. FN plays a role in non-neoplasmatic lung pathology in which fibronectin gene polymorphisms (RFLPs) have been studied. The aim of our work was to evaluate the frequency of two of fibronectin RFLPs: genotypes AB, AA, BB (HaeIII) and CD, CC, DD (MspI) in patients with lung cancer. The studied group consisted of 63 patients with squamous cell lung cancer and 53 controls without any malignant or proliferative disease. There were no statistically significant differences in the distribution of studied genotypes between lung cancer patients and controls.

GD1a Overcomes Inhibition of Myelination by Fibronectin via Activation of Protein Kinase A: Implications for Multiple Sclerosis.

PubMed

Qin, Jing; Sikkema, Arend H; van der Bij, Kristine; de Jonge, Jenny C; Klappe, Karin; Nies, Vera; Jonker, Johan W; Kok, Jan Willem; Hoekstra, Dick; Baron, Wia

2017-10-11

Remyelination failure by oligodendrocytes contributes to the functional impairment that characterizes the demyelinating disease multiple sclerosis (MS). Since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting remyelination are pivotal in halting disease progression. Our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure by perturbing oligodendrocyte progenitor cell (OPC) maturation. Here, we aim at elucidating whether exogenously added gangliosides (i.e., cell surface lipids with a potential to modulate signaling pathways) could counteract fibronectin-mediated inhibition of OPC maturation. Exclusive exposure of rat oligodendrocytes to GD1a, but not other gangliosides, overcomes aggregated fibronectin-induced inhibition of myelin membrane formation, in vitro , and OPC differentiation in fibronectin aggregate containing cuprizone-induced demyelinated lesions in male mice. GD1a exerts its effect on OPCs by inducing their proliferation and, at a late stage, by modulating OPC maturation. Kinase activity profiling revealed that GD1a activated a protein kinase A (PKA)-dependent signaling pathway and increased phosphorylation of the transcription factor cAMP response element-binding protein. Consistently, the effect of GD1a in restoring myelin membrane formation in the presence of fibronectin aggregates was abolished by the PKA inhibitor H89, whereas the effect of GD1a was mimicked by the PKA activator dibutyryl-cAMP. Together, GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation by activating a PKA-dependent signaling pathway. Given the persistent presence of fibronectin aggregates in MS lesions, ganglioside GD1a might act as a potential novel therapeutic tool to selectively modulate the detrimental signaling environment that precludes remyelination. SIGNIFICANCE STATEMENT As an environmental factor, aggregates of the extracellular matrix protein fibronectin perturb the maturation of oligodendrocyte progenitor cells (OPCs), thereby impeding remyelination, in the demyelinating disease multiple sclerosis (MS). Here we demonstrate that exogenous addition of ganglioside GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation, both in vitro and in vivo , by activating a PKA-dependent signaling pathway. We propose that targeted delivery of GD1a to MS lesions may act as a potential novel molecular tool to boost maturation of resident OPCs to overcome remyelination failure and halt disease progression. Copyright © 2017 the authors 0270-6474/17/379925-14$15.00/0.

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers.

PubMed

De, Pradip; Carlson, Jennifer H; Wu, Hui; Marcus, Adam; Leyland-Jones, Brian; Dey, Nandini

2016-07-12

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronectin-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.

Function of actin cytoskeleton in gravisensing during spaceflight

NASA Astrophysics Data System (ADS)

Hughes-Fulford, M.

Since astronauts and cosmonauts have significant bone loss in microgravity, we hypothesized that there would be physiological changes in cellular bone growth in the absence of gravity. Our first experiments on STS-56 demonstrated that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) and a paradoxical 2 fold increase in release of autocrine PGE2 when compared to ground controls. In addition, there was a significant collapse of the actin cytoskeleton and loss of focal adhesions after 4 days of growth in microgravity. Other investigators have made similar observations of cytoskeletal modifications in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the cytoskeleton in cells grown in microgravity conditions, however flown cells under 1g conditions maintained normal actin cytoskeleton and fibronectin matrix. We do not think that the changes seen in the cytoskeleton are due to alterations in fibronectin message or protein synthesis since no differences were found between microgravity, 1g or ground conditions. The nuclear structure was noticeably different in the flown 0g cells with elongation of the nucleus after 24 hours of microgravity, this alteration in nuclear structure was not seen in the 1g flown or ground control cells. Further examination of total RNA in the cells showed no significant changes between the three gravity conditions suggesting specific not general physiological changes in microgravity. When osteoblast mRNA was analyzed, the immediate early genes, c-myc and cox-2 and the autocrine growth factor FGFb were down-regulated in microgravity. The inability of the 0g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways. It is still unclear whether these changes in signal transduction are related to the alterations in the cytoskeleton under microgravity conditions and this possibility is under study.

A novel mechanism of UV-A and riboflavin-mediated corneal cross-linking through induction of tissue transglutaminases.

PubMed

Kopsachilis, Nikolaos; Tsaousis, Konstantinos T; Tsinopoulos, Ioannis T; Kruse, Friedrich E; Welge-Luessen, Ulrich

2013-07-01

Collagen cross-linking using UV-A irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. The purposes of this study were to examine whether primary human corneal keratocytes (HCKs) are capable of expressing and secreting fibronectin and tissue transglutaminase (tTgase), an enzyme cross-linking extracellular matrix protein, and to examine whether fibronectin and tTgase are increased after the treatment of HCK cells with UV-A irradiation combined with riboflavin (RFUV-A), thus providing another possible physiological mechanism of the cross-linking pathway. Cell cultures established from HCKs were treated with 0.025% riboflavin solution and UV-A (370 nm) irradiance 3 mW/cm2 for 30 minutes. Induction of fibronectin and tTgase was investigated by immunohistochemistry, real-time polymerase chain reaction, and Western blot analysis. Cell viability was quantified by a microscopic live-dead assay. External tTgase activity was measured by the ability to form polymerized fibronectin and the incorporation of biotinylated cadaverine into fibronectin. Treatment of cultured HCK cells with RFUV-A increased the fibronectin and tTgase messenger RNA and protein levels. This effect was not observed in cells treated with riboflavin or UV-A radiation alone. Incorporation of biotinylated cadaverine was significantly increased when HCK cells were treated with RFUV-A. The enzymes tTgase and fibronectin are expressed by RFUV-A treatment in cultured HCK cells. This mechanism provides more information about the physiology of corneal cross-linking.

Predictive value of cervical length measurement and fibronectin testing in threatened preterm labor.

PubMed

van Baaren, Gert-Jan; Vis, Jolande Y; Wilms, Femke F; Oudijk, Martijn A; Kwee, Anneke; Porath, Martina M; Oei, Guid; Scheepers, Hubertina C J; Spaanderman, Marc E A; Bloemenkamp, Kitty W M; Haak, Monique C; Bolte, Antoinette C; Bax, Caroline J; Cornette, Jérôme M J; Duvekot, Johannes J; Nij Bijvanck, Bas W A; van Eyck, Jim; Franssen, Maureen T M; Sollie, Krystyna M; Vandenbussche, Frank P H A; Woiski, Mallory; Grobman, William A; van der Post, Joris A M; Bossuyt, Patrick M M; Opmeer, Brent C; Mol, Ben W J

2014-06-01

To estimate the performance of combining cervical length measurement with fetal fibronectin testing in predicting delivery in women with symptoms of preterm labor. We conducted a prospective nationwide cohort study in all 10 perinatal centers in The Netherlands. Women with symptoms of preterm labor between 24 and 34 weeks of gestation with intact membranes were included. In all women, qualitative fibronectin testing (0.050-microgram/mL cutoff) and cervical length measurement were performed. Logistic regression was used to predict spontaneous preterm delivery within 7 days after testing. A risk less than 5%, corresponding to the risk for women with a cervical length of at least 25 mm, was considered as low risk. Between December 2009 and August 2012, 714 women were enrolled. Fibronectin results and cervical length were available for 665 women, of whom 80 (12%) delivered within 7 days. Women with a cervical length of at least 30 mm or with a cervical length between 15 and 30 mm with a negative fibronectin result were at low risk (less than 5%) of spontaneous delivery within 7 days. Fibronectin testing in case of a cervical length between 15 and 30 mm additionally classified 103 women (15% of the cohort) as low risk and 36 women (5% of the cohort) as high risk. Cervical length measurement, combined with fetal fibronectin testing in case of a cervical length between 15 and 30 mm, improves identification of women with a low risk to deliver spontaneously within 7 days. II.

Promotion of osteoblast differentiation in 3D biomaterial micro-chip arrays comprising fibronectin-coated poly(methyl methacrylate) polycarbonate.

PubMed

Altmann, Brigitte; Steinberg, Thorsten; Giselbrecht, Stefan; Gottwald, Eric; Tomakidi, Pascal; Bächle-Haas, Maria; Kohal, Ralf-Joachim

2011-12-01

Due to the architecture of solid body tissues including bone, three-dimensional (3D) in vitro microenvironments appear favorable, since herein cell growth proceeds under more physiological conditions compared to conventional 2D systems. In the present study we show that a 3D microenvironment comprising a fibronectin-coated PMMA/PC-based micro-chip promotes differentiation of primary human osteoblasts as reflected by the densely-packed 3D bone cell aggregates and expression of biomarkers indicating osteoblast differentiation. Morphogenesis and fluorescence dye-based live/dead staining revealed homogenous cell coverage of the microcavities of the chip array, whereat cells showed high viability up to 14 days. Moreover, Azur II staining proved formation of uniform sized multilayered aggregates, exhibiting progressive intracellular deposition of extracellular bone matrix constituents comprising fibronectin, osteocalcin and osteonectin from day 7 on. Compared to 2D monolayers, osteoblasts grown in the 3D chip environment displayed differential mostly higher gene expression for osteocalcin, osteonectin, and alkaline phosphatase, while collagen type I remained fairly constant in both culture environments. Our results indicate that the 3D microenvironment, based on the PMMA biomaterial chip array promotes osteoblast differentiation, and hereby renders a promising tool for tissue-specific in vitro preconditioning of osteoblasts designated for clinically-oriented bone augmentation or regeneration. Copyright © 2011 Elsevier Ltd. All rights reserved.

Nanometer-sized extracellular matrix coating on polymer-based scaffold for tissue engineering applications.

PubMed

Uchida, Noriyuki; Sivaraman, Srikanth; Amoroso, Nicholas J; Wagner, William R; Nishiguchi, Akihiro; Matsusaki, Michiya; Akashi, Mitsuru; Nagatomi, Jiro

2016-01-01

Surface modification can play a crucial role in enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering applications. Here, we report a novel approach for layer-by-layer (LbL) fabrication of nanometer-size fibronectin and gelatin (FN-G) layers on electrospun fibrous poly(carbonate urethane)urea (PCUU) scaffolds. Alternate immersions into the solutions of fibronectin and gelatin provided thickness-controlled FN-G nano-layers (PCUU(FN-G) ) which maintained the scaffold's 3D structure and width of fibrous bundle of PCUU as evidenced by scanning electron miscroscopy. The PCUU(FN-G) scaffold improved cell adhesion and proliferation of bladder smooth muscles (BSMCs) when compared to uncoated PCUU. The high affinity of PCUU(FN-G) for cells was further demonstrated by migration of adherent BSMCs from culture plates to the scaffold. Moreover, the culture of UROtsa cells, human urothelium-derived cell line, on PCUU(FN-G) resulted in an 11-15 μm thick multilayered cell structure with cell-to-cell contacts although many UROtsa cells died without forming cell connections on PCUU. Together these results indicate that this approach will aid in advancing the technology for engineering bladder tissues in vitro. Because FN-G nano-layers formation is based on nonspecific physical adsorption of fibronectin onto polymer and its subsequent interactions with gelatin, this technique may be applicable to other polymer-based scaffold systems for various tissue engineering/regenerative medicine applications. © 2015 Wiley Periodicals, Inc.

Placental Alpha Microglobulin-1 Compared With Fetal Fibronectin to Predict Preterm Delivery in Symptomatic Women.

PubMed

Wing, Deborah A; Haeri, Sina; Silber, Angela C; Roth, Cheryl K; Weiner, Carl P; Echebiri, Nelson C; Franco, Albert; Pappas, Lanissa M; Yeast, John D; Brebnor, Angelle A; Quirk, J Gerald; Murphy, Aisling M; Laurent, Louise C; Field, Nancy T; Norton, Mary E

2017-12-01

To compare the rapid bedside test for placental α microglobulin-1 with the instrumented fetal fibronectin test for prediction of imminent spontaneous preterm delivery among women with symptoms of preterm labor. We conducted a prospective observational study on pregnant women with signs or symptoms suggestive of preterm labor between 24 and 35 weeks of gestation with intact membranes and cervical dilatation less than 3 cm. Participants were prospectively enrolled at 15 U.S. academic and community centers. Placental α microglobulin-1 samples did not require a speculum examination. Health care providers were blinded to placental α microglobulin-1 results. Fetal fibronectin samples were collected through speculum examination per manufacturer requirements and sent to a certified laboratory for testing using a cutoff of 50 ng/mL. The coprimary endpoints were positive predictive value (PPV) superiority and negative predictive value (NPV) noninferiority of placental α microglobulin-1 compared with fetal fibronectin for the prediction of spontaneous preterm birth within 7 days and within 14 days. Of 796 women included in the study cohort, 711 (89.3%) had both placental α microglobulin-1 and fetal fibronectin results and valid delivery outcomes available for analysis. The overall rate of preterm birth was 2.4% (17/711) within 7 days of testing and 4.2% (30/711) within 14 days of testing with respective rates of spontaneous preterm birth of 1.3% (9/703) and 2.9% (20/701). Fetal fibronectin was detected in 15.5% (110/711), and placental α microglobulin-1 was detected in 2.4% (17/711). The PPVs for spontaneous preterm delivery within 7 days or less among singleton gestations (n=13) for placental α microglobulin-1 and fetal fibronectin were 23.1% (3/13) and 4.3% (4/94), respectively (P<.025 for superiority). The NPVs were 99.5% (619/622) and 99.6% (539/541) for placental α microglobulin-1 and fetal fibronectin, respectively (P<.001 for noninferiority). Although placental α microglobulin-1 performed the same as fetal fibronectin in ruling out spontaneous preterm delivery among symptomatic women, it demonstrated statistical superiority in predicting it.

Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis.

PubMed

Jafari, Abbas; Qanie, Diyako; Andersen, Thomas L; Zhang, Yuxi; Chen, Li; Postert, Benno; Parsons, Stuart; Ditzel, Nicholas; Khosla, Sundeep; Johansen, Harald Thidemann; Kjærsgaard-Andersen, Per; Delaisse, Jean-Marie; Abdallah, Basem M; Hesselson, Daniel; Solberg, Rigmor; Kassem, Moustapha

2017-02-14

Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A Biosynthetic Nerve Guide Conduit Based on Silk/SWNT/Fibronectin Nanocomposite for Peripheral Nerve Regeneration

PubMed Central

Mottaghitalab, Fatemeh; Farokhi, Mehdi; Zaminy, Arash; Kokabi, Mehrdad; Soleimani, Masoud; Mirahmadi, Fereshteh

2013-01-01

As a contribution to the functionality of nerve guide conduits (NGCs) in nerve tissue engineering, here we report a conduit processing technique through introduction and evaluation of topographical, physical and chemical cues. Porous structure of NGCs based on freeze-dried silk/single walled carbon nanotubes (SF/SWNTs) has shown a uniform chemical and physical structure with suitable electrical conductivity. Moreover, fibronectin (FN) containing nanofibers within the structure of SF/SWNT conduits produced through electrospinning process have shown aligned fashion with appropriate porosity and diameter. Moreover, fibronectin remained its bioactivity and influenced the adhesion and growth of U373 cell lines. The conduits were then implanted to 10 mm left sciatic nerve defects in rats. The histological assessment has shown that nerve regeneration has taken places in proximal region of implanted nerve after 5 weeks following surgery. Furthermore, nerve conduction velocities (NCV) and more myelinated axons were observed in SF/SWNT and SF/SWNT/FN groups after 5 weeks post implantation, indicating a functional recovery for the injured nerves. With immunohistochemistry, the higher S-100 expression of Schwann cells in SF/SWNT/FN conduits in comparison to other groups was confirmed. In conclusion, an oriented conduit of biocompatible SF/SWNT/FN has been fabricated with acceptable structure that is particularly applicable in nerve grafts. PMID:24098649

Fabrication of functional fibronectin patterns by nanosecond excimer laser direct write for tissue engineering applications.

PubMed

Grigorescu, S; Hindié, M; Axente, E; Carreiras, F; Anselme, K; Werckmann, J; Mihailescu, I N; Gallet, O

2013-07-01

Laser direct write techniques represent a prospective alternative for engineering a new generation of hybrid biomaterials via the creation of patterns consisting of biological proteins onto practically any type of substrate. In this paper we report on the characterization of fibronectin features obtained onto titanium substrates by UV nanosecond laser transfer. Fourier-transform infrared spectroscopy measurements evidenced no modification in the secondary structure of the post-transferred protein. The molecular weight of the transferred protein was identical to the initial fibronectin, no fragment bands being found in the transferred protein's Western blot migration profile. The presence of the cell-binding domain sequence and the mannose groups within the transferred molecules was revealed by anti-fibronectin monoclonal antibody immunolabelling and FITC-Concanavalin-A staining, respectively. The in vitro tests performed with MC3T3-E1 osteoblast-like cells and Swiss-3T3 fibroblasts showed that the cells' morphology and spreading were strongly influenced by the presence of the fibronectin spots.

Novel mechanism of transcriptional regulation of cell matrix protein through CREB

PubMed Central

Habib, Samy L; Mohan, Sumathy; Liang, Sitai; Li, Baojie; Yadav, Mukesh

2015-01-01

The transcription mechanism(s) of renal cell matrix accumulation in diabetes does not explored. Phosphorylation of the transcription factor cAMP-responsive element binding protein (CREB) significantly increased in cells treated with high glucose (HG) compared to cell grown in normal glucose (NG). Cells pretreated with rapamycin before exposure to HG showed significant decrease phosphorylation of CREB, increase in AMPK activity and decrease protein/mRNA and promoter activity of fibronectin. In addition, cells transfected with siRNA against CREB showed significant increase in AMPK activity, decrease in protein/mRNA and promoter activity of fibronectin. Cells treated with HG showed nuclear localization of p-CREB while pretreated cells with rapamycin reversed HG effect. Moreover, gel shift analysis shows increase binding of CREB to fibronectin promoter in cells treated with HG while cells pretreated with rapamycin reversed the effect of HG. Furthermore, db/db mice treated with rapamycin showed significant increase in AMPK activity, decrease in expression of p-CREB and protein/mRNA of fibronectin. Strong staining of fibronectin and p-CREB was detected in kidney cortex of db/db mice while treated mice with rapamycin reversed hyperglycemia effect. In summary, our data provide a novel mechanism of transcriptional regulation of fibronectin through CREB that may be used as therapeutic approach to prevent diabetes complications. PMID:26115221

Fibronectin regulates the activation of THP-1 cells by TGF-beta1.

PubMed

Wang, A C; Fu, L

2001-03-01

To determine how fibronectin regulates the immunomodulatory effects of transforming growth factor (TGF)-beta on THP-1 cells. THP-1 monocytic cell line. THP-1 cells were primed for 48 h in the presence or absence of 250 pM TGF-beta1. Assays or assessments carried out, together with statistical test applied. We found that adherence to fibronectin dramatically modulates the effects of TGF-beta1 on the human monocytic cell line THP-1. TGF-beta did not significantly affect constitutive interleukin (IL)-8 secretion or IL-1beta-induced IL-8 secretion from suspended cells. In contrast, TGF-beta stimulated IL-8 secretion as well as augmented IL-1beta-induced IL-8 secretion from adherent cells. The differential effects of TGF-beta1 on IL-8 secretion from suspended and adherent cells could not be explained by differences in IL-1 receptor antagonist production. The effects of fibronectin on TGF-beta1 induced IL-8 secretion from THP-1 cells were mimicked by adhesion to immobilized anti-a4beta1 integrin antibody and to a fibronectin fragment containing the CS-1 domain. These results indicate that alpha4beta1-mediated adhesion to fibronectin may play a key role during inflammation by profoundly influencing the effects of TGF-beta1 on monocytes.

Osseoconductivity of a Specific Streptavidin-Biotin-Fibronectin Surface Coating of Biotinylated Titanium Implants - A Rabbit Animal Study.

PubMed

Kämmerer, Peer W; Lehnert, Michael; Al-Nawas, Bilal; Kumar, Vinay V; Hagmann, Sebastien; Alshihri, Abdulmonem; Frerich, Bernhard; Veith, Michael

2015-10-01

Biofunctionalized implant surfaces may accelerate bony integration and increase long-term stability. The aim of the study was to evaluate the osseous reaction toward biomimetic titanium implants surfaces coated with quasicovalent immobilized fibronectin in an in vivo animal model. A total of 84 implants (uncoated [control 1, n = 36], streptavidin-biotin coated [test 1, n = 24], streptavidin-biotin-fibronectin coated [test 2, n = 24]) were inserted 1 mm supracortically in the proximal tibia of 12 rabbits. The samples were examined after 3 and 6 weeks. Total bone-implant contact (tBIC; %), bone-implant contact in the cortical (cBIC; %) and in the spongious bone (sBIC; %) as well as the percentage of linear bone fill (PLF; %) were evaluated. After 3 weeks, streptavidin-biotin-fibronectin implants had a significant higher sBIC (p = .043) and PLF (p = .007) compared with the uncoated samples. After 6 weeks, this difference was significant for tBIC (p = .016) and cBIC (p < .001). Additionally, uncoated screws showed a significant higher sBIC when compared with the fibronectin coating (p < .001). Streptavidin-biotin-coated implants showed less bone growth at both time points of all examined parameters when compared with their counterparts (all p < .001). Quasicovalent immobilization of biotinylated fibronectin with the streptavidin-biotin-fibronectin system on smooth surface titanium shows a beneficial faster osseous healing in vivo. Besides, an antifouling effect of the streptavidin-biotin coating was proven. © 2015 Wiley Periodicals, Inc.

Connection between integrins and cell activation in rat adrenal glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of the p42/p44(mapk) pathway and intracellular calcium.

PubMed

Campbell, Shirley; Otis, Melissa; Côté, Mylène; Gallo-Payet, Nicole; Payet, Marcel Daniel

2003-04-01

Integrins are responsible for adhesion and activation of several intracellular cascades. The present study was aimed at determining whether the interaction between fibronectin and integrins could generate pathways involved in physiological functions of rat adrenal glomerulosa cells. Immunofluorescence studies and adhesion assays showed that fibronectin was the best matrix in promoting the formation of focal adhesion. Binding of glomerulosa cells to fibronectin, but not to collagen I or poly-L-lysine, involved the integrin-binding sequence Arg-Gly-Asp (RGD). Activation of glomerulosa cells with Arg-Gly-Asp-Ser (RGDS) induced an increase in [Ca(2+)](i), whereas fibronectin triggered a release of Ca(2+) from InsP(3)-sensitive Ca(2+) stores. Aldosterone secretion induced by ACTH, angiotensin II, and RGDS and proliferation were improved on fibronectin, compared with poly-L-lysine. The RGDS peptide induced a transient increase in the activity of the p42/p44(mapk), independent of phosphatidylinositol-3 kinase and protein kinase C. Integrins alpha(5) and alpha(V) as well as their fibronectin receptor partners beta(1) and beta(3), were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the alpha(5)beta(1), alpha(v)beta(1), or alpha(v)beta(3) integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44(mapk) cascade, leading to cell proliferation and aldosterone secretion.

The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus.

PubMed

Danger, Jessica L; Makthal, Nishanth; Kumaraswami, Muthiah; Sumby, Paul

2015-12-01

The group A Streptococcus (GAS; Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to the prtF1 and prtF2 mRNAs within their 5' untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and the prtF1 and prtF2 mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection. More than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small RNA FasX. In addition to identifying that FasX reduces the abundance of the cell surface-located fibronectin-binding proteins PrtF1/2, fibronectin is present in high abundance in human tissues, and we have determined the mechanism behind this regulation. Importantly, as FasX is the only mechanistically characterized regulatory RNA in GAS, it serves as a model RNA in this and related pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Biochemical basis of vocal fold mobilization after microflap surgery in a rabbit model.

PubMed

Mitchell, Joshua R; Kojima, Tsuyoshi; Wu, Hongmei; Garrett, C Gaelyn; Rousseau, Bernard

2014-02-01

To investigate phonation-related extracellular matrix (ECM) changes in the vocal fold lamina propria after microflap surgery using an in vivo rabbit phonation model. Prospective animal study. Twenty-four New Zealand White rabbits were used in this study. Quantitative polymerase chain reaction and immunohistochemistry were used to investigate alterations in vocal fold ECM proinflammatory and profibrotic gene, and protein expression from a control group of animals receiving a microflap without phonation and a separate group of animals receiving experimentally induced phonation on postmicroflap days 0, 3, and 7. IHC demonstrated the highest concentration of CD45 in vocal folds on postoperative day 0. Staining for CD45 was absent by postoperative day 7, with no differences in CD45 staining between groups. Fibronectin gene expression increased significantly on postoperative day 3 in the control and experimentally induced phonation groups, with maximal staining of fibronectin around the microflap incision on postoperative day 7. No alterations in cyclooxygenase-2, interleukin-1β, and transforming growth factor-β1 gene expression were observed between groups. Results of the present study revealed an acute inflammatory response in the vocal fold at the time of microflap (day 0) and up to 3 days post-microflap. By post-operative day 3, staining of CD45 positive cells decreased, with essentially no evidence of inflammation by post-operative day 7. With the end of the acute inflammatory response occurring around day 3, these data may provide support for mobilizing tissue after inflammation has subsided and the process of active tissue remodeling has ensued (days 3-7). N/A. © 2013 The American Laryngological, Rhinological and Otological Society, Inc.

Impedimetric Analysis of the Effect of Decellularized Porcine Heart Scaffold on Human Fibrosarcoma, Endothelial, and Cardiomyocyte Cell Lines

PubMed Central

Bäcker, Henrik; Polgár, Livia; Soós, Pal; Lajkó, Eszter; Láng, Orsolya; Merkely, Bela; Szabó, Gabor; Dohmen, Pascal M.; Weymann, Alexander; Kőhidai, Laszlo

2017-01-01

Background Experiments on porcine heart scaffold represent significant assays in development of immunoneutral materials for cardiac surgery. Characterization of cell-cell and cell-scaffold interactions is essential to understand the homing process of cardiac cells into the scaffolds. Material/Methods In the present study, the highly sensitive and real-time impedimetric technique of xCELLigence SP was used to monitor cell adhesion, which is the key process of recellularization in heart scaffolds. Our objectives were: (i) to characterize the effect of decellularized porcine heart scaffold on cell adhesion of human cardiovascular cells potentially used in the recellularization process; and (ii) to investigate cell-extracellular matrix element interactions for building artificial multi-layer systems, applied as cellular models of recellularization experiments. Human fibrosarcoma, endothelial, and cardiomyocyte cells were investigated and the effect of decellularized porcine heart scaffold (HS) and fibronectin on cell adhesion was examined. Adhesion was quantified as slope of curves. Results Heart scaffold had neutral effect on cardiomyocytes as well as on endothelial cells. Adhesion of cardiomyocytes was increased by fibronectin (1.480±0.021) compared to control (0.745±0.029). The combination of fibronectin and HS induced stronger adhesion of cardiomyocytes (2.407±0.634) than fibronectin alone. Endothelial and fibrosarcoma cells showed similarly strong adhesion profiles with marked enhancer effect by fibronectin. Conclusions Decellularized porcine HS does not inhibit adhesion of human cardiovascular cells at the cell biological level, while fibronectin has strong cell adhesion-inducer effect, as well as an enhancer effect on activity of HS. Consequently, decellularized porcine hearts could be used as scaffolds for recellularization with cardiomyocytes and endothelial cells with fibronectin acting as a regulator, leading to construction of working bioartificial hearts. PMID:28493851

Endothelin and sex hormones modulate the fibronectin synthesis by cultured human skin scleroderma fibroblasts

PubMed Central

Soldano, S; Montagna, P; Villaggio, B; Parodi, A; Gianotti, G; Sulli, A; Seriolo, B; Secchi, M E; Cutolo, M

2009-01-01

Objective: To evaluate the influence of endothelin-1 (ET-1) and sex hormones on cell proliferation and extracellular matrix (ECM) synthesis (ie, fibronectin, laminin) by cultured normal and scleroderma (SSc) human skin fibroblasts (FBs). Methods: Primary cultures of FBs were treated with ET-1 and sex hormones (17β-oestradiol or testosterone) for 24 h. Cell growth was analysed by methiltetrazolium salt test, ECM synthesis was evaluated by immunocytochemistry and western blot, both at 24 h. Results: In normal FBs, ET-1 and 17β-oestradiol, as well as their combination, increased cell growth (p<0.001, p<0.001, p<0.01 vs untreated cells (control), respectively) and fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). By contrast, testosterone either alone or in combination with ET-1 did not influence cell proliferation, but decreased fibronectin synthesis (p<0.05, testosterone vs control). In SSc FBs, ET-1 and 17β-oestradiol alone or their combination induced an increased fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). Unexpectedly, testosterone induced an increase of fibronectin synthesis (p<0.05 vs control). Conclusions: ET-1 and 17β-oestradiol seem to exert a profibrotic effect in normal and SSc culture FBs and might suggest their synergistic effect in the pathogenesis of the fibrotic process in SSc. PMID:18952637

Chemically grafted fibronectin for use in QCM-D cell studies

PubMed Central

Sobolewski, Peter; Tomczyk, Nancy; Composto, Russell J.; Eckmann, David M.

2014-01-01

Traditionally, fibronectin has been used as a physisorbed surface coating (physFN) in cell culture experiments due to its critical role in cell adhesion. However, because the resulting layer is thick, unstable, and of unpredictable uniformity, this method of fibronectin deposition is unsuitable for some types of research, including quartz crystal microbalance (QCM) experiments involving cells. Here, we present a new method for chemical immobilization of fibronectin onto silicon oxide surfaces, including QCM crystals pre-coated with silicon oxide. We characterize these chemically coated fibronectin surfaces (chemFN) as well as physFN ones using surface ellipsometry (SE), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. A cell culture model demonstrates that cells on chemFN and physFN surfaces exhibit similar viability, structure, adhesion and metabolism. Finally, we perform QCM experiments using cells on both surfaces which demonstrate the superior suitability of chemFN coatings for QCM research, and provide real-time QCM-D data from cells subjected to an actin depolymerizing agent. Overall, our method of chemical immobilization of fibronectin yields great potential for furthering cellular experiments in which thin, stable and uniform coatings are desirable. As QCM research with cells has been rather limited in success thus far, we anticipate that this new technique will particularly benefit this experimental system by availing it to the much broader field of cell mechanics. PMID:24657645

FOXC1 Regulates Expression of Prostaglandin Receptors Leading to an Attenuated Response to Latanoprost.

PubMed

Doucette, Lance P; Footz, Tim; Walter, Michael A

2018-05-01

This study examines the effect of FOXC1 on the prostaglandin pathway in order to explore FOXC1's role in the prostaglandin-resistant glaucoma phenotype commonly seen in Axenfeld-Rieger syndrome. Binding and transcriptional activity of FOXC1 to the gene coding for the EP3 prostaglandin receptor (PTGER3) were evaluated through ChIP-qPCR and luciferase-based assays. Immortalized trabecular meshwork cells (TM1) and HeLa cells had FOXC1 mRNA reduced via siRNA interference. qPCR and Western blot experiments were conducted to examine the changes in prostaglandin receptor expression brought about by lowered FOXC1. TM1 cells were then treated with 10 μM latanoprost acid and/or an siRNA for FOXC1. The expression of fibronectin and matrix metalloproteinase 9 were evaluated via qPCR in each treatment condition. ChIP-qPCR and luciferase experiments confirmed that FOXC1 binds to and activates transcription of the EP3 gene prostaglandin receptor. qPCR and Western experiments in HeLa and TM1 cells showed that FOXC1 siRNA knockdown results in significantly lowered EP3 levels (protein and RNA). In addition, RNA levels of the other prostaglandin receptor genes EP1 (PTGER1), EP2 (PTGER2), EP4 (PTGER4), and FP (PTGFR) were altered when FOXC1 was knocked down in TM1 and HeLa cells. Analysis of fibronectin expression in TM1 cells after treatment with 10 μM latanoprost acid showed a statistically significant increase in expression; this increase was abrogated by cotreatment with a siRNA for FOXC1. We show the abrogation of latanoprost signalling when FOXC1 is knocked down via siRNA in a trabecular meshwork cell line. We propose that the lower levels of active FOXC1 in Axenfeld-Rieger syndrome patients with glaucoma account for the lack of response to prostaglandin-based medications.

The Haemophilus influenzae Hap Autotransporter Binds to Fibronectin, Laminin, and Collagen IV

PubMed Central

Fink, Doran L.; Green, Bruce A.; St. Geme III, Joseph W.

2002-01-01

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Haps, from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Haps. Purified Haps binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Haps to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract. PMID:12183535

Silk-based biomaterials functionalized with fibronectin type II promotes cell adhesion.

PubMed

Pereira, Ana Margarida; Machado, Raul; da Costa, André; Ribeiro, Artur; Collins, Tony; Gomes, Andreia C; Leonor, Isabel B; Kaplan, David L; Reis, Rui L; Casal, Margarida

2017-01-01

The objective of this work was to exploit the fibronectin type II (FNII) module from human matrix metalloproteinase-2 as a functional domain for the development of silk-based biopolymer blends that display enhanced cell adhesion properties. The DNA sequence of spider dragline silk protein (6mer) was genetically fused with the FNII coding sequence and expressed in Escherichia coli. The chimeric protein 6mer+FNII was purified by non-chromatographic methods. Films prepared from 6mer+FNII by solvent casting promoted only limited cell adhesion of human skin fibroblasts. However, the performance of the material in terms of cell adhesion was significantly improved when 6mer+FNII was combined with a silk-elastin-like protein in a concentration-dependent behavior. With this work we describe a novel class of biopolymer that promote cell adhesion and potentially useful as biomaterials for tissue engineering and regenerative medicine. This work reports the development of biocompatible silk-based composites with enhanced cell adhesion properties suitable for biomedical applications in regenerative medicine. The biocomposites were produced by combining a genetically engineered silk-elastin-like protein with a genetically engineered spider-silk-based polypeptide carrying the three domains of the fibronectin type II module from human metalloproteinase-2. These composites were processed into free-standing films by solvent casting and characterized for their biological behavior. To our knowledge this is the first report of the exploitation of all three FNII domains as a functional domain for the development of bioinspired materials with improved biological performance. The present study highlights the potential of using genetically engineered protein-based composites as a platform for the development of new bioinspired biomaterials. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Dysregulated fibronectin trafficking by Hsp90 inhibition restricts prostate cancer cell invasion.

PubMed

Armstrong, Heather K; Gillis, Joanna L; Johnson, Ian R D; Nassar, Zeyad D; Moldovan, Max; Levrier, Claire; Sadowski, Martin C; Chin, Mei Yieng; Tomlinson Guns, Emma S; Tarulli, Gerard; Lynn, David J; Brooks, Douglas A; Selth, Luke A; Centenera, Margaret M; Butler, Lisa M

2018-02-01

The molecular chaperone Hsp90 is overexpressed in prostate cancer (PCa) and is responsible for the folding, stabilization and maturation of multiple oncoproteins, which are implicated in PCa progression. Compared to first-in-class Hsp90 inhibitors such as 17-allylamino-demethoxygeldanamycin (17-AAG) that were clinically ineffective, second generation inhibitor AUY922 has greater solubility and efficacy. Here, transcriptomic and proteomic analyses of patient-derived PCa explants identified cytoskeletal organization as highly enriched with AUY922 treatment. Validation in PCa cell lines revealed that AUY922 caused marked alterations to cell morphology, and suppressed cell motility and invasion compared to vehicle or 17-AAG, concomitant with dysregulation of key extracellular matrix proteins such as fibronectin (FN1). Interestingly, while the expression of FN1 was increased by AUY922, FN1 secretion was significantly decreased. This resulted in cytosolic accumulation of FN1 protein within late endosomes, suggesting that AUY922 disrupts vesicular secretory trafficking pathways. Depletion of FN1 by siRNA knockdown markedly reduced the invasive capacity of PCa cells, phenocopying AUY922. These results highlight a novel mechanism of action for AUY922 beyond its established effects on cellular mitosis and survival and, furthermore, identifies extracellular matrix cargo delivery as a potential therapeutic target for the treatment of aggressive PCa.

Adipose progenitor cells increase fibronectin matrix strain and unfolding in breast tumors

NASA Astrophysics Data System (ADS)

Chandler, E. M.; Saunders, M. P.; Yoon, C. J.; Gourdon, D.; Fischbach, C.

2011-02-01

Increased stiffness represents a hallmark of breast cancer that has been attributed to the altered physicochemical properties of the extracellular matrix (ECM). However, the role of fibronectin (Fn) in modulating the composition and mechanical properties of the tumor-associated ECM remains unclear. We have utilized a combination of biochemical and physical science tools to evaluate whether paracrine signaling between breast cancer cells and adipose progenitor cells regulates Fn matrix assembly and stiffness enhancement in the tumor stroma. In particular, we utilized fluorescence resonance energy transfer imaging to map the molecular conformation and stiffness of Fn that has been assembled by 3T3-L1 preadipocytes in response to conditioned media from MDA-MB231 breast cancer cells. Our results reveal that soluble factors secreted by tumor cells promote Fn expression, unfolding, and stiffening by adipose progenitor cells and that transforming growth factor-β serves as a soluble cue underlying these changes. In vivo experiments using orthotopic co-transplantation of primary human adipose-derived stem cells and MDA-MB231 into SCID mice support the pathological relevance of our results. Insights gained by these studies advance our understanding of the role of Fn in mammary tumorigenesis and may ultimately lead to improved anti-cancer therapies.

Aliskiren Regulates Neonatal Fc Receptor and IgG Metabolism with Attenuation of Anti-GBM Glomerulonephritis in Mice.

PubMed

Kang, Ju Hyung; Baik, Haing Woon; Yoo, Seung-Min; Kim, Joo Heon; Cheong, Hae Il; Park, Chung-Gyu; Kang, Hee Gyung; Ha, Il-Soo

2016-01-01

Renin, in addition to its activation of the renin-angiotensin system, binds to the (pro)renin receptor (PRR) and triggers inflammatory and fibrogenic signaling in tissue. In addition, aliskiren, a direct renin inhibitor, has been shown to affect IgG metabolism by altering PRR and neonatal Fc receptors (FcRns). We investigated the effect of aliskiren on proteinuria, glomerular extracellular matrix, expressions of fibronectin, transforming growth factor β1 (TGF-β1), PRR, FcRn and renal metabolism of IgG in a mice model of anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). IgG deposition and expressions of FcRn and PRR were enhanced at glomeruli and urinary IgG levels increased in anti-GBM GN. Aliskiren attenuated anti-GBM GN with reduction of proteinuria and cortical expressions of fibronectin and TGF-β1. In addition, aliskiren suppressed the renal cortical expressions of FcRn and PRR. Aliskiren also reduced the glomerular IgG depositions and the urinary IgG levels albeit with increased circulating serum IgG levels. These results suggest that suppression of FcRn and PRR and regulation of IgG metabolism may be related to the attenuation of anti-GBM GN by aliskiren. © 2016 S. Karger AG, Basel.

Osteoblast adhesion to orthopaedic implant alloys: Effects of cell adhesion molecules and diamond-like carbon coating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kornu, R.; Kelly, M.A.; Smith, R.L.

1996-11-01

In total joint arthroplasty, long-term outcomes depend in part on the biocompatibility of implant alloys. This study analyzed effects of surface finish and diamond-like carbon coating on osteoblast cell adhesion to polished titanium-aluminum-vanadium and polished or grit-blasted cobalt-chromium-molybdenum alloys. Osteoblast binding was tested in the presence and absence of the cell adhesion proteins fibronectin, laminin, fibrinogen, and vitronectin and was quantified by measurement of DNA content. Although adherence occurred in serum-free medium, maximal osteoblast binding required serum and was similar for titanium and cobalt alloys at 2 and 12 hours. With the grit-blasted cobalt alloy, cell binding was reduced 48%more » (p < 0.05) by 24 hours. Coating the alloys with diamond-like carbon did not alter osteoblast adhesion, whereas fibronectin pretreatment increased cell binding 2.6-fold (p < 0.05). In contrast, fibrinogen, vitronectin, and laminin did not enhance cell adhesion. These results support the hypothesis that cell adhesion proteins can modify cell binding to orthopaedic alloys. Although osteoblast binding was not affected by the presence of diamond-like carbon, this coating substance may influence other longer term processes, such as bone formation, and deserves further study. 40 refs., 4 figs.« less

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas.

PubMed

Shimoyama, S; Gansauge, F; Gansauge, S; Oohara, T; Beger, H G

1995-12-01

The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine alpha-subunits (alpha 2-alpha 6, alpha V, alpha L, alpha M, and alpha X), four beta-subunits (beta 1, beta 3-beta 5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except alpha V showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed alpha V. Whereas alpha 2, alpha 3, and alpha 6 expression was stronger and diffuse, no alpha 5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between alpha 2, alpha 3, and alpha 6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.

Phenotypic changes in neutrophils related to anti-inflammatory therapy.

PubMed

Barton, A E; Bayley, D L; Mikami, M; Llewellyn-Jones, C G; Stockley, R A

2000-01-03

Previous work from the group has shown that non-steroidal anti-inflammatory agents given to volunteers and patients inhibit PMN function possibly by affecting the developing neutrophil during the differentiation process. In this study indomethacin treatment in vivo reduced neutrophil chemotaxis and proteolytic degradation of fibronectin, with a maximal effect after 14 days. Stimulated neutrophil adherence to fibronectin was also reduced but this was not due to quantitative changes in beta(2) integrin expression or function. L-Selectin expression on resting and stimulated neutrophils was increased after 14 days and there was a small decrease in plasma levels of soluble L-selectin. These effects, however, could not be reproduced by treatment of neutrophils with indomethacin in vitro, suggesting they are due to effects on differentiating/maturing PMNs. In an attempt to interpret these changes, studies were performed with dexamethasone, which is known to alter neutrophil function and kinetics. Dexamethasone treatment reduced chemotaxis and increased superoxide generation after 1 day and was associated with increased expression of activated beta(2) integrins and reduced L-selectin expression on resting neutrophils. This suggests the appearance of mainly 'activated' cells as a result of demargination and indicates that the effects of indomethacin are distinctive and not related to changes in compartmentalisation.

Advances in our understanding of the Reinke space.

PubMed

Thibeault, Susan L

2005-06-01

Normal vocal fold vibration depends critically upon the composition of the Reinke space or the lamina propria extracellular matrix. Alterations in the normal composition of the extracellular matrix result in a loss of normal vibratory function. In this article, the present literature on the Reinke space in normal and disease states is reviewed including publications in the multidisciplinary fields of biomechanics, histology, molecular biology, and tissue engineering. With recent technology advances, the etiology for benign lesions has been investigated with computer models and bioreactors. Particular extracellular matrix constituents in various benign vocal fold lesions--fibronectin, fibromodulin and hyaluronan--appear to be involved in altering the viscoelastic properties of the Reinke space. Significant basic science approaches to the investigation of the characterization of the Reinke space in vocal fold scarring has produced several potential future treatment avenues. Tissue-engineering approaches for regeneration of the Reinke space are the most recent addition to the literature showing promising research directions. Voice disorders represent a significant clinical problem. Research attempting to discover the underlying molecular and genetic regulation and homeostasis of the extracellular matrix of the Reinke space are essential. Effective future clinical interventions must be based upon the knowledge of how genetic and biologic features are disturbed in vocal diseases and how they relate to vocal symptoms.

Flagellin and GroEL mediates in vitro binding of an atypical enteropathogenic Escherichia coli to cellular fibronectin.

PubMed

Moraes, Claudia T P; Polatto, Juliana M; Rossato, Sarita S; Izquierdo, Mariana; Munhoz, Danielle D; Martins, Fernando H; Pimenta, Daniel C; Farfan, Mauricio J; Elias, Waldir P; Barbosa, Ângela S; Piazza, Roxane M F

2015-12-18

Enteropathogenic Escherichia coli (EPEC) is distinguished mainly by the presence of EPEC adherence factor plasmid (pEAF) in typical EPEC (tEPEC) and its absence in atypical EPEC (aEPEC). The initial adherence to the intestinal mucosa is complex and mediated by adhesins other than bundle-forming pilus, which is not produced by aEPEC. Extracellular matrix (ECM) proteins of eukaryotic cells are commonly recognized by bacterial adhesins. Therefore, binding to ECM proteins may facilitate colonization, invasion and/or signaling by intestinal pathogens. Previous studies from our group demonstrated that aEPEC O26:H11 (strain BA2103) showed high binding activity to fibronectin, not shared by its counterpart, aEPEC O26:HNM. In the present study, using mass spectrometry after fibronectin-associated immunoprecipitation, two proteins, flagellin (50 kDa) and GroEL (52 kDa), were identified and BA2103 binding ability to fibronectin was inhibited in the presence of anti-H11 and anti-GroEL sera, but not by either naïve rabbit or other unrelated sera. It was also observed that the presence of purified flagellin inhibits adhesion of BA2103 to cellular fibronectin in a dose-dependent manner. Additionally, BA2103 GroEL is similar to the same protein of uropathogenic E. coli. Our results suggest that flagellin may play a role in the in vitro interaction of BA2103 with cellular fibronectin, and GroEL can be an accessory protein in this process.

Polymorphisms in Fibronectin Binding Protein A of Staphylococcus Aureus are Associated with Infection of Cardiovascular Devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lower, Steven; Lamlertthon, Supaporn; Casillas-Ituarte, Nadia

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a bio-film, a structured community of bacterial cells adherent to the surface of a solid substrate. Every bio-film begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated frommore » humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct bindingforce signature and had speci!c single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.« less

Oleic acid-induced ANGPTL4 enhances head and neck squamous cell carcinoma anoikis resistance and metastasis via up-regulation of fibronectin.

PubMed

Shen, Chih-Jie; Chan, Shih-Hung; Lee, Chung-Ta; Huang, Wan-Chen; Tsai, Jhih-Peng; Chen, Ben-Kuen

2017-02-01

Obese patients have higher levels of free fatty acids (FFAs) in their plasma and a higher risk of cancer than their non-obese counterparts. However, the mechanisms involved in the regulation of cancer metastasis by FFAs remain unclear. In this study, we found that oleic acid (OA) induced angiopoietin-like 4 (ANGPTL4) protein expression and secretion and conferred anoikis resistance to head and neck squamous cell carcinomas (HNSCCs). The autocrine production of OA-induced ANGPTL4 further promoted HNSCC migration and invasion. In addition, the expression of peroxisome proliferator-activated receptor (PPAR) was essential for the OA-induced ANGPTL4 expression and invasion. The levels of OA-induced epithelial-mesenchymal transition markers, such as vimentin, MMP-9, and fibronectin and its downstream effectors Rac1/Cdc42, were significantly reduced in ANGPTL4-depleted cells. Knocking down fibronectin inhibited the expression of MMP-9 and repressed OA- and recombinant ANGPTL4-induced HNSCC invasion. On the other hand, ANGPTL4 siRNA inhibited OA-induced MMP-9 expression, which was reversed in fibronectin-overexpressing cells. Furthermore, the depletion of ANGPTL4 impeded the OA-primed metastatic seeding of tumor cells in the lungs. These results demonstrate that OA enhances HNSCC metastasis through the ANGPTL4/fibronectin/Rac1/Cdc42 and ANGPTL4/fibronectin/MMP-9 signaling axes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Biopolymers and supramolecular polymers as biomaterials for biomedical applications

PubMed Central

Freeman, Ronit; Boekhoven, Job; Dickerson, Matthew B.; Naik, Rajesh R.

2015-01-01

Protein- and peptide-based structural biopolymers are abundant building blocks of biological systems. Either in their natural forms, such as collagen, silk or fibronectin, or as related synthetic materials they can be used in various technologies. An emerging area is that of biomimetic materials inspired by protein-based biopolymers, which are made up of small molecules rather than macromolecules and can therefore be described as supramolecular polymers. These materials are very useful in biomedical applications because of their ability to imitate the extracellular matrix both in architecture and their capacity to signal cells. This article describes important features of the natural extracellular matrix and highlight how these features are being incorporated into biomaterials composed of biopolymers and supramolecular polymers. We particularly focus on the structures, properties, and functions of collagen, fibronectin, silk, and the supramolecular polymers inspired by them as biomaterials for regenerative medicine. PMID:26989295

αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin

PubMed Central

Bharadwaj, Mitasha; Strohmeyer, Nico; Colo, Georgina P.; Helenius, Jonne; Beerenwinkel, Niko; Schiller, Herbert B.; Fässler, Reinhard; Müller, Daniel J.

2017-01-01

Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5β1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5β1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5β1 integrins. Once engaged, αV-class integrins signal to α5β1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5β1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix. PMID:28128308

A low-molecular-weight galactofucan from the seaweed, Spatoglossum schröederi, binds fibronectin and inhibits capillary-like tube formation in vitro.

PubMed

Menezes, Maira Maria; Nobre, Leonardo Thiago Duarte Barreto; Rossi, Gustavo Rodrigues; Almeida-Lima, Jailma; Melo-Silveira, Raniere Fagundes; Franco, Celia Regina Cavichiolo; Trindade, Edvaldo Silva; Nader, Helena Bonciani; Rocha, Hugo Alexandre Oliveira

2018-05-01

A low-molecular-weight (LMW) heterofucan (designated fucan B) was obtained from the brown seaweed, Spatoglossum schröederi, and its activity as an inhibitor of capillary-like tube formation by endothelial cells (ECs) was analyzed. Chemical, infrared and electrophoretic analyses confirmed the identity of fucan B. In contrast to other LMW fucans, fucan B (0.012-0.1 mg/mL) inhibited ECs capillary-like tube formation in a concentration-dependent manner. In addition, fucan B (0.01-0.05 mg/mL) did not affect ECs proliferation. Fucan B also inhibited ECs migration on a fibronectin-coated surface, but not on laminin- or collagen-coated surfaces. Biotinylated fucan B was used as a probe to identify its localization. Confocal microscopy experiments revealed that biotinylated fucan did not bind to the cell surface, but rather only to fibronectin. Our findings suggest that fucan B inhibits ECs capillary-like tube formation and migration by binding directly to fibronectin and blocking fibronectin sites recognized by cell surface ligands. However, further studies are needed to evaluate the in vivo effects of fucan B. Copyright © 2018 Elsevier B.V. All rights reserved.

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells.

PubMed

Lee, Hyung-Ok; Mullins, Stefanie R; Franco-Barraza, Janusz; Valianou, Matthildi; Cukierman, Edna; Cheng, Jonathan D

2011-06-13

Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated. Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions. © 2011 Lee et al; licensee BioMed Central Ltd.

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

PubMed Central

2011-01-01

Background Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. Methods We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. Results We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated. Conclusion Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions. PMID:21668992

Gene editing of the extra domain A positive fibronectin in various tumors, amplified the effects of CRISPR/Cas system on the inhibition of tumor progression.

PubMed

Lv, Wan-Qi; Wang, Hai-Cheng; Peng, Jing; Wang, Yi-Xiang; Jiang, Jiu-Hui; Li, Cui-Ying

2017-12-01

The low efficiency of clustered, regularly interspaced, palindromic repeats-associated Cas (CRISPR/Cas) system editing genes in vivo limits the application. A components of the extracellular matrix (ECM), the extra domain A positive fibronectin (EDA+FN), may be a target for CRISPR/Cas system for the pro-oncogenic effects. The exclusion of EDA exon would alter the microenvironment and inhibit tumor progression, even the frequency of gene editing is still limited. The pro-oncogenic effects were confirmed by the exclusion of EDA exon from the fibronectin gene, as illustrated by the down-regulated proliferation, migration and invasion of CNE-2Z or SW480 cells (P<0.05). Furthermore, although the efficacy of EDA exon knockout through CRISPR/Cas system was shown to be low in vivo , the EDA+FN protein levels decrease obviously, inhibiting the tumor growth rate significantly (P<0.05), which was accompanied by a decrease in Ki-67 expression and microvessel numbers, and increased E-cadherin or decreased Vimentin expression (P<0.05). Human nasopharyngeal carcinoma cell line CNE-2Z, and the colorectal carcinoma cell line SW480 were transfected with CRISPR/Cas9 plasmids targeting EDA exon. The effects of the exclusion of EDA on the cell proliferation, motility and epithelial-mesenchymal transition (EMT) were investigated, and the western blot and real-time PCR were performed to analyze the underlying mechanisms. Furthermore, CRISPR/Cas9 plasmids were injected into xenograft tumors to knockout EDA exon in vivo , and tumor growth, cell proliferation, EMT rate, or vascularization were investigated using western blot, PCR and immunohistochemistry. CRISPR/Cas system targeting ECM components was shown to be an effective method for the inhibition of tumor progression, as these paracrine or autocrine molecules are necessary for various tumor cells. This may represent a novel strategy for overcoming the drug evasion or resistance, in addition, circumventing the low efficiency of CRISPR/Cas system in vivo .

Acellularization-Induced Changes in Tensile Properties Are Organ Specific - An In-Vitro Mechanical and Structural Analysis of Porcine Soft Tissues

PubMed Central

Aust, Gabriela; Boldt, Andreas; Fritsch, Sebastian; Keil, Isabel; Koch, Holger; Möbius, Robert; Scheidt, Holger A.; Wagner, Martin F. X.; Hammer, Niels

2016-01-01

Introduction Though xenogeneic acellular scaffolds are frequently used for surgical reconstruction, knowledge of their mechanical properties is lacking. This study compared the mechanical, histological and ultrastructural properties of various native and acellular specimens. Materials and Methods Porcine esophagi, ureters and skin were tested mechanically in a native or acellular condition, focusing on the elastic modulus, ultimate tensile stress and maximum strain. The testing protocol for soft tissues was standardized, including the adaption of the tissue’s water content and partial plastination to minimize material slippage as well as templates for normed sample dimensions and precise cross-section measurements. The native and acellular tissues were compared at the microscopic and ultrastructural level with a focus on type I collagens. Results Increased elastic modulus and ultimate tensile stress values were quantified in acellular esophagi and ureters compared to the native condition. In contrast, these values were strongly decreased in the skin after acellularization. Acellularization-related decreases in maximum strain were found in all tissues. Type I collagens were well-preserved in these samples; however, clotting and a loss of cross-linking type I collagens was observed ultrastructurally. Elastins and fibronectins were preserved in the esophagi and ureters. A loss of the epidermal layer and decreased fibronectin content was present in the skin. Discussion Acellularization induces changes in the tensile properties of soft tissues. Some of these changes appear to be organ specific. Loss of cross-linking type I collagen may indicate increased mechanical strength due to decreasing transverse forces acting upon the scaffolds, whereas fibronectin loss may be related to decreased load-bearing capacity. Potentially, the alterations in tissue mechanics are linked to organ function and to the interplay of cells and the extracellular matrix, which is different in hollow organs when compared to skin. PMID:26960134

HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

PubMed

González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

2017-10-10

The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.

Osteopontin and fibronectin levels are decreased in vitreous of autoimmune uveitis and retinal expression of both proteins indicates ECM re-modeling.

PubMed

Deeg, Cornelia A; Eberhardt, Christina; Hofmaier, Florian; Amann, Barbara; Hauck, Stefanie M

2011-01-01

Autoimmune uveitis is an intraocular inflammation that arises through autoreactive T-cells attacking the inner eye, eventually leading to blindness. However, the contributing molecular pathomechanisms within the affected tissues remain as yet elusive. The extracellular matrix (ECM) is a highly dynamic structure that varies tremendously and influences the encompassing tissue. In order to assess ECM re-modeling in autoimmune uveitis, we investigated the expression of ECM molecules fibronectin and osteopontin in vitreous and retina samples. This was carried out in the only spontaneous animal model for human autoimmue uveitis, namely equine recurrent uveitis (ERU) that resembles the human disease in clinical as well as in immunopathological aspects. ERU is a naturally occurring autoimmune disease in horses that develops frequently and has already proved its value to study disease-related pathomechanisms. Western blot analysis of fibronectin and osteopontin in healthy and uveitic vitreous revealed significant reduction of both proteins in uveitis. Immunohistochemical expression of fibronectin in healthy retinas was restricted to the inner limiting membrane abutting vimentin positive Müller cell endfeet, while in uveitic sections, a disintegration of the ILM was observed changing the fibronectin expression to a dispersed pattern extending toward the vitreous. Retinal expression of osteopontin in control tissue was found in a characteristic Müller cell pattern illustrated by co-localization with vimentin. In uveitic retinas, the immunoreactivity of osteopontin in gliotic Müller cells was almost absent. The ability of Müller cells to express fibronectin and osteopontin was additionally shown by immunocytochemistry of primary cultured equine Müller cells and the equine Müller cell line eqMC-7. In conclusion, severe ECM re-modeling in autoimmune uveitis reported here, might affect the adhesive function of fibronectin and thus the anchoring of Müller cell endfeet to the ILM. Furthermore, the absence of osteopontin in gliotic Müller cells might represent reduced neuroprotection, an osteopontin attribute that is intensively discussed.

Platelet-rich plasma and chronic wounds: remaining fibronectin may influence matrix remodeling and regeneration success.

PubMed

Moroz, Andrei; Deffune, Elenice

2013-11-01

Platelet-rich plasma has been largely used as a therapeutic option for the treatment of chronic wounds of different etiologies. The enhanced regeneration observed after the use of platelet-rich plasma has been systematically attributed to the growth factors that are present inside platelets' granules. We hypothesize that the remaining plasma and platelet-bound fibronectin may act as a further bioactive protein in platelet-rich plasma preparations. Recent reports were analyzed and presented as direct evidences of this hypotheses. Fibronectin may directly influence the extracellular matrix remodeling during wound repair. This effect is probably through matrix metalloproteinase expression, thus exerting an extra effect on chronic wound regeneration. Physicians should be well aware of the possible fibronectin-induced effects in their future endeavors with PRP in chronic wound treatment. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

PDGF-A suppresses contact inhibition during directional collective cell migration.

PubMed

Nagel, Martina; Winklbauer, Rudolf

2018-06-08

The leading edge mesendoderm (LEM) of the Xenopus gastrula moves as an aggregate by collective migration. However, LEM cells on fibronectin in vitro show contact inhibition of locomotion by quickly retracting lamellipodia upon mutual contact. We found that a fibronectin-integrin-syndecan module acts between p21-activated kinase-1 upstream and ephrinB1 downstream to promote the contact-induced collapse of lamellipodia. To function in this module, fibronectin has to be present as puncta on the surface of LEM cells. To overcome contact inhibition in LEM cell aggregates, PDGF-A deposited in the endogenous substratum of LEM migration blocks the fibronectin-integrin-syndecan module at the integrin level. This stabilizes lamellipodia preferentially in the direction of normal LEM movement and supports cell orientation and the directional migration of the coherent LEM cell mass. © 2018. Published by The Company of Biologists Ltd.

Single-molecule dynamic force spectroscopy of the fibronectin-heparin interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Gabriel; Lamontagne, Charles-Antoine; Lebel, Rejean

2007-12-21

The integrity of cohesive tissues strongly depends on the presence of the extracellular matrix, which provides support and anchorage for cells. The fibronectin protein and the heparin-like glycosaminoglycans are key components of this dynamic structural network. In this report, atomic force spectroscopy was used to gain insight into the compliance and the resistance of the fibronectin-heparin interaction. We found that this interaction can be described by an energetic barrier width of 3.1 {+-} 0.2 A and an off-rate of 0.2 {+-} 0.1 s{sup -1}. These dissociation parameters are similar to those of other carbohydrate-protein interactions and to off-rate values reportedmore » for more complex interactions between cells and extracellular matrix components. Our results indicate that the function of the fibronectin-heparin interaction is supported by its capacity to sustain significant deformations and considerable external mechanical forces.« less

Function of the cytoskeleton in gravisensing during spaceflight

NASA Astrophysics Data System (ADS)

Hughes-Fulford, M.

2003-10-01

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-folf increase in release of the osteoblast autocrine factor PGE 2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE 2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the Og grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block cell growth either in G1 (F-actin microfilament collapse), or in G2/M (inhibition of microtubule polymerization during G2/M-phase). We therefore hypothesize that microgravity would inhibit growth in either G1, or G2/M.

Upregulation of Fibronectin and the α5β1 and αvβ3 Integrins on Blood Vessels within the Cerebral Ischemic Penumbra

PubMed Central

Li, Longxuan; Liu, Fudong; Welser-Alves, Jennifer V.; McCullough, Louise D.; Milner, Richard

2012-01-01

Following focal cerebral ischemia, blood vessels in the ischemic border, or penumbra, launch an angiogenic response. In light of the critical role for fibronectin in angiogenesis, and the observation that fibronectin and its integrin receptors are strongly upregulated on angiogenic vessels in the hypoxic CNS, the aim of this study was to establish whether angiogenic vessels in the ischemic CNS also show this response. Focal cerebral ischemia was established in C57/Bl6 mice by middle cerebral artery occlusion (MCA:O), and brain tissue analyzed seven days following re-perfusion, a time at which angiogenesis is ongoing. Within the ischemic core, immunofluorescent (IF) studies demonstrated vascular expression of MECA-32, a marker of leaky cerebral vessels, and vascular breakdown, defined by loss of staining for the endothelial marker, CD31, and the vascular adhesion molecules, laminin, dystroglycan and α6 integrin. Within the ischemic penumbra, dual-IF with CD31 and Ki67 revealed the presence of proliferating endothelial cells, indicating ongoing angiogenesis. Significantly, vessels in the ischemic penumbra showed strong upregulation of fibronectin and the fibronectin receptors, α5β1 and αvβ3 integrins. Taken together with our recent finding that the α5β1 integrin plays an important role in promoting cerebral angiogenesis in response to hypoxia, these results suggest that stimulation of the fibronectin-α5β1 integrin signalling pathway may provide a novel approach to amplifying the intrinsic angiogenic response to cerebral ischemia. PMID:22056225

Leptospira santorosai Serovar Shermani detergent extract induces an increase in fibronectin production through a Toll-like receptor 2-mediated pathway.

PubMed

Tian, Ya-Chung; Hung, Cheng-Chieh; Li, Yi-Jung; Chen, Yung-Chang; Chang, Ming-Yang; Yen, Tzung-Hai; Hsu, Hsiang-Hao; Wu, Mai-Szu; Phillips, Aled; Yang, Chih-Wei

2011-03-01

Leptospirosis can activate inflammatory responses through Toll-like receptors (TLRs) and may cause renal tubulointerstitial fibrosis characterized by the accumulation of extracellular matrix (ECM). We have previously demonstrated that Leptospira santorosai serovar Shermani detergent extract stimulates ECM accumulation in vitro. The aim of this study was to examine the mechanistic basis of these previous observations and, in particular, to examine the potential involvement of TLRs. The addition of serovar Shermani detergent extract led to an increase in fibronectin gene expression and production. Inhibition of TLR2 but not TLR4 expression abrogated serovar Shermani detergent extract-mediated increases in fibronectin production. This response was also blocked by the knockdown of the gene expression of the TLR2 downstream transducers myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). Serovar Shermani detergent extract also activated nuclear factor-κB, and its inhibition by curcumin-attenuated serovar Shermani detergent extract induced increases in fibronectin production. These effects were also mimicked by the specific TLR2 agonist, Pam(3)CsK(4), a response that was also abrogated by the knockdown of MyD88 and TRAF6. Similarly, the administration of live leptospires to cells also induced fibronectin production that was blocked by inhibition of TLR2 and MyD88 expression. In conclusion, serovar Shermani detergent extract can induce fibronectin production through the TLR2-associated cascade, providing evidence of an association between TLRs and leptospirosis-mediated ECM deposition.

Quantitative fetal fibronectin and cervical length to predict preterm birth in asymptomatic women with previous cervical surgery.

PubMed

Vandermolen, Brooke I; Hezelgrave, Natasha L; Smout, Elizabeth M; Abbott, Danielle S; Seed, Paul T; Shennan, Andrew H

2016-10-01

Quantitative fetal fibronectin testing has demonstrated accuracy for prediction of spontaneous preterm birth in asymptomatic women with a history of preterm birth. Predictive accuracy in women with previous cervical surgery (a potentially different risk mechanism) is not known. We sought to compare the predictive accuracy of cervicovaginal fluid quantitative fetal fibronectin and cervical length testing in asymptomatic women with previous cervical surgery to that in women with 1 previous preterm birth. We conducted a prospective blinded secondary analysis of a larger observational study of cervicovaginal fluid quantitative fetal fibronectin concentration in asymptomatic women measured with a Hologic 10Q system (Hologic, Marlborough, MA). Prediction of spontaneous preterm birth (<30, <34, and <37 weeks) with cervicovaginal fluid quantitative fetal fibronectin concentration in primiparous women who had undergone at least 1 invasive cervical procedure (n = 473) was compared with prediction in women who had previous spontaneous preterm birth, preterm prelabor rupture of membranes, or late miscarriage (n = 821). Relationship with cervical length was explored. The rate of spontaneous preterm birth <34 weeks in the cervical surgery group was 3% compared with 9% in previous spontaneous preterm birth group. Receiver operating characteristic curves comparing quantitative fetal fibronectin for prediction at all 3 gestational end points were comparable between the cervical surgery and previous spontaneous preterm birth groups (34 weeks: area under the curve, 0.78 [95% confidence interval 0.64-0.93] vs 0.71 [95% confidence interval 0.64-0.78]; P = .39). Prediction of spontaneous preterm birth using cervical length compared with quantitative fetal fibronectin for prediction of preterm birth <34 weeks of gestation offered similar prediction (area under the curve, 0.88 [95% confidence interval 0.79-0.96] vs 0.77 [95% confidence interval 0.62-0.92], P = .12 in the cervical surgery group; and 0.77 [95% confidence interval 0.70-0.84] vs 0.74 [95% confidence interval 0.67-0.81], P = .32 in the previous spontaneous preterm birth group). Prediction of spontaneous preterm birth using cervicovaginal fluid quantitative fetal fibronectin in asymptomatic women with cervical surgery is valid, and has comparative accuracy to that in women with a history of spontaneous preterm birth. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanisms of Mechano-Transduction Within Osteoblasts

DTIC Science & Technology

2001-09-01

bone sialoprotein , and fibronectin) that are the ligands for these receptors. We propose that the expression of these proteins is regulated in...system(s) that are responsible for mediating osteopontin, bone sialoprotein and fibronectin gene expression in response to mechanical stimulation, will

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

PubMed

Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Hyaluronic acid (with fibronectin) as a bioimplant for the vocal fold mucosa.

PubMed

Chan, R W; Titze, I R

1999-07-01

To measure the viscoelastic shear properties of hyaluronic acid, with and without fibronectin, and to compare them with those of the human vocal fold mucosa and other phonosurgical biomaterials. Viscoelastic shear properties of various implantable biomaterials (Teflon, gelatin, collagen, fat, hyaluronic acid, and hyaluronic acid with fibronectin) were measured with a parallel-plate rotational rheometer. Elastic and viscous shear properties were quantified as a function of oscillation frequency (0.01-15 Hz) at 37 degrees C. The shear properties of hyaluronic acid were relatively close to those of human vocal fold mucosal tissues reported previously. Hyaluronic acid at specific concentrations (0.5%-1%), with or without fibronectin, was found to exhibit viscous shear properties (viscous shear modulus and dynamic viscosity) similar to those of the average male and female vocal fold mucosa. According to a theory that establishes the effects of tissue shear properties on vocal fold oscillation, phonation threshold pressure (a measure of the ease of phonation) is directly related to the viscous shear modulus of the vibrating vocal fold mucosa. Therefore, our findings suggest that hyaluronic acid, either by itself or mixed with fibronectin, may be a potentially optimal bioimplant for the surgical management of vocal fold mucosal defects and lamina propria deficiencies (e.g., scarring) from a biomechanical standpoint.

Complementary effects of multi-protein components on biomineralization in vitro

PubMed Central

Ba, Xiaolan; Rafailovich, Miriam; Meng, Yizhi; Pernodet, Nadine; Wirick, Sue; Füredi-Milhofer, Helga; Qin, Yi-Xian; DiMasi, Elaine

2016-01-01

The extracellular matrix (ECM) is composed of mixed protein fibers whose precise composition affects biomineralization. New methods are needed to probe the interactions of these proteins with calcium phosphate mineral and with each other. Here we follow calcium phosphate mineralization on protein fibers self-assembled in vitro from solutions of fibronectin, elastin and their mixture. We probe the surface morphology and mechanical properties of the protein fibers during the early stages. The development of mineral crystals on the protein matrices is also investigated. In physiological mineralization solution, the elastic modulus of the fibers in the fibronectin-elastin mixture increases to a greater extent than that of the fibers from either pure protein. In the presence of fibronectin, longer exposure in the mineral solution leads to the formation of amorphous calcium phosphate particles templated along the self-assembled fibers, while elastin fibers only collect calcium without any mineral observed during early stage. TEM images confirm that small needle-shape crystals are confined inside elastin fibers which suppress the release of mineral outside the fibers during late stage, while hydroxyapatite crystals form when fibronectin is present. These results demonstrate complementary actions of the two ECM proteins fibronectin and elastin to collect cations and template mineral, respectively. PMID:20035875

Hypertrophic scar contracture is mediated by the TRPC3 mechanical force transducer via NFkB activation

PubMed Central

Ishise, Hisako; Larson, Barrett; Hirata, Yutaka; Fujiwara, Toshihiro; Nishimoto, Soh; Kubo, Tateki; Matsuda, Ken; Kanazawa, Shigeyuki; Sotsuka, Yohei; Fujita, Kazutoshi; Kakibuchi, Masao; Kawai, Kenichiro

2015-01-01

Wound healing process is a complex and highly orchestrated process that ultimately results in the formation of scar tissue. Hypertrophic scar contracture is considered to be a pathologic and exaggerated wound healing response that is known to be triggered by repetitive mechanical forces. We now show that Transient Receptor Potential (TRP) C3 regulates the expression of fibronectin, a key regulatory molecule involved in the wound healing process, in response to mechanical strain via the NFkB pathway. TRPC3 is highly expressed in human hypertrophic scar tissue and mechanical stimuli are known to upregulate TRPC3 expression in human skin fibroblasts in vitro. TRPC3 overexpressing fibroblasts subjected to repetitive stretching forces showed robust expression levels of fibronectin. Furthermore, mechanical stretching of TRPC3 overexpressing fibroblasts induced the activation of nuclear factor-kappa B (NFκB), a regulator fibronectin expression, which was able to be attenuated by pharmacologic blockade of either TRPC3 or NFκB. Finally, transplantation of TRPC3 overexpressing fibroblasts into mice promoted wound contraction and increased fibronectin levels in vivo. These observations demonstrate that mechanical stretching drives fibronectin expression via the TRPC3-NFkB axis, leading to intractable wound contracture. This model explains how mechanical strain on cutaneous wounds might contribute to pathologic scarring. PMID:26108359

Adhesion of a monolayer of fibroblast cells to fibronectin under sonic vibrations in a bioreactor.

PubMed

Titze, Ingo R; Klemuk, Sarah A; Lu, Xiaoying

2012-06-01

We examined cell adhesion to a surface under vibrational forces approximating those of phonation. A monolayer of human fibroblast cells was seeded on a fibronectin-coated glass coverslip, which was attached to either the rotating part or the stationary part of a rheometer-bioreactor. The temperature, humidity, carbon dioxide level, nutrients, and cell seeding density were controlled. The cell density was on the order of 1,000 to 5,000 cells per square millimeter. Target stresses above 1 kPa at an oscillatory frequency of 100 Hz were chosen to reflect conditions of vocal fold tissue vibration. Fibronectin coating provided enough adhesion to support at least 2 kPa of oscillating stress, but only about 0.1 kPa of steady rotational shear. For stresses exceeding those limits, the cells were not able to adhere to the thin film of fibronectin. Cells will adhere to a planar surface under stresses typical of phonation, which provide a more stringent test than adherence in a 3-dimensional matrix. The density of cell seeding on the coverslip played a role in cell-extracellular matrix adhesion, in that the cells adhered to each other more than to the fibronectin coating when the cells were nearly confluent.

The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

PubMed

Smelter, Dan F; de Lange, Willem J; Cai, Wenxuan; Ge, Ying; Ralphe, J Carter

2018-06-01

Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of haploinsufficiency. NEW & NOTEWORTHY This study is one of the first to describe a disease mechanism for a missense mutation in cardiac myosin-binding protein C linked to hypertrophic cardiomyopathy. The mutation decreases stability of the fibronectin type III domain and results in substantially reduced mutant protein expression dissonant to transcript abundance.

Chondroitin sulphates A, B and C, collagen types I-IV and fibronectin in venous sinus of the red pulp in human spleen.

PubMed

Rovenská, E; Michalka, P; Papincák, J; Durdík, S; Jakubovský, J

2005-01-01

The morphological relationship of chondroitin sulphates A, B, and C, collagen types I-IV and fibronectin in the wall of venous sinuses of the red pulp in human spleen has not been a focus of interest among morphologists. Regarding the hypothesis that the structure of the spleen lends it the function of a blood filter the substances described in our study might play a significant role in the functional morphology. Of 146 human spleen surgical specimens, groups of 12 specimens each were examined under a light microscope using the method of antibodies against fibronectin, against collagen types I-IV and against chondroitin sulphates A, B, and C. The sections of the red pulp of human spleen stained with hematoxylin and eosin provided limited information about the wall of the sinuses. Chondroitin sulphates A and B were observed on the surface of sinus-lining cells (SLC), and fibronectin was detected on the surface of the annular fibers. Collagen type 11 was observed almost in the same places as chondroitin sulphates A and B. Collagen type IV was present in annular fibers of the wall of the sinus and in the basement membrane, like fibronectin. Chondroitin sulphate was not present in the walls of sinuses. Binding of antibodies against chondroitin sulphate A and against chondroitin sulphate B indicates the presence of chondroitin sulfates on the surface of SLC, where they probably play a role in helping the human organism to recognize alien and self substances. The presence of chondroitin,sulphates A and B probably affects inhibition of binding of cells with collagen type I, but not with fibronectin.

Immunohistochemical study of integrin α₅β₁, fibronectin, and Bcl-2 in normal oral mucosa, inflammatory fibroepithelial hyperplasia, oral epithelial dysplasia, and oral squamous cell carcinoma.

PubMed

Núñez, Manuel Antonio Gordón; de Matos, Felipe Rodrigues; Freitas, Roseana de Almeida; Galvão, Hébel Cavalcanti

2013-07-01

The objective of this study was to compare the immunoexpression of integrin α₅β₁, fibronectin, and the Bcl-2 protein in normal oral mucosa (NOM), inflammatory fibroepithelial hyperplasia (IFH), oral epithelial dysplasia (OED), and oral squamous cell carcinoma (OSCC). Eleven cases of NOM, 16 IFH, 20 OED, and 27 OSCC were selected for analysis of the immunoexpression of integrin α₅β₁, fibronectin, and bcl-2 protein. There was an association between the intensity and location of the integrin α₅β₁ expression, especially in the OSCC, that 48.1% of cases showed weak immunoreactivity and 40.7% in the suprabasal layer (P < 0.05). There was an association between the pattern and distribution of fibronectin expression in basement membrane, where 90% of NOM showed a pattern of linear continuous and 80% of OED exhibited focal distribution (P < 0.05). The fibronectin expression in connective tissue was predominantly intense with an association of staining pattern among the different specimens, where 37% of OSCC showed a reticular pattern (P < 0.05). There was an association of bcl-2 protein among the types of specimens, especially in IFH and OSCC, where 100% of the cases exhibited scores 1 of staining (P < 0.05). Within this context, the interaction of integrin α₅β₁ with its main ligand in the extracellular matrix, fibronectin, is suggested to influence the survival of tumor cells and to favor their proliferation by modulating apoptosis through the upregulation of antiapoptotic proteins or the suppression of apoptotic mediators.

Distinct Effects of RGD-glycoproteins on Integrin-Mediated Adhesion and Osteogenic Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Schwab, Elisabeth H.; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A.

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α5-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β3-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation. PMID:24324361

Distinct effects of RGD-glycoproteins on Integrin-mediated adhesion and osteogenic differentiation of human mesenchymal stem cells.

PubMed

Schwab, Elisabeth H; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α₅-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β₃-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.

Tissue distribution of very late activation antigens-1/6 and very late activation antigen ligands in the normal thymus and in thymoma.

PubMed Central

Ruco, L. P.; Paradiso, P.; Pittiglio, M.; Diodoro, M. G.; Gearing, A. J.; Mainiero, F.; Gismondi, A.; Santoni, A.; Baroni, C. D.

1993-01-01

The expression of very late activation antigens (VLAs)-1/6 was correlated with that of the VLA ligands fibronectin, laminin, collagen, and vascular cell adhesion molecule-1 in sections of normal thymus, in thymocyte suspensions, and in 10 cases of thymoma. Capsular epithelial cells are VLA-2+, VLA-3+, and VLA-6+ and face the thymic basement membrane, which is rich in fibronectin, laminin, and collagen type IV. Cortical epithelial cells are VLA-2+ and are embedded in a reticular meshwork of nonorganized extracellular matrix (ECM) that is rich in fibronectin. Cortical thymocytes, identified as CD3dim cells by using immunofluorescence in suspension, are highly positive for VLA-4, a fibronectin ligand. Most cortical macrophages are positive for vascular cell adhesion molecule-1, a molecule recognized by VLA-4. Medullary epithelial cells are VLA-2+/VLA-3+ and are codistributed with fibrous strands of organized ECM that are positive for fibronectin, collagen, and laminin. Medullary thymocytes, identified as CD3bright cells, are positive for VLA-4 and VLA-6, a ligand for laminin. Our findings suggest that intrathymic thymocyte maturation is associated with changes in expression of VLA molecules, which are apparently correlated with the presence of VLA ligands in the tissue microenvironment. Thymomas were classified as cortical (three), common (five), or medullary (two) type. Expression of VLA molecules and distribution of ECM in the three histological subtypes were reminiscent of those observed in the respective regions of the normal thymus. All cases of thymoma were characterized by overexpression of VLA molecules on neoplastic cells, which was associated with increased deposition of organized ECM rich in fibronectin, laminin, and collagen. Images Figure 1 Figure 3 PMID:8456937

CLONING, EXPRESSION, AND HEMOSTATIC ACTIVITIES OF A DISINTEGRIN, r-MOJASTIN 1, FROM THE MOHAVE RATTLESNAKE (Crotalus scutulatus scutulatus)

PubMed Central

Sánchez, Elda E.; Lucena, Sara E.; Reyes, Steven; Soto, Julio G.; Cantu, Esteban; Lopez-Johnston, Juan Carlos; Guerrero, Belsy; Salazar, Ana Maria; Rodríguez-Acosta, Alexis; Galán, Jacob A.; Tao, W. Andy; Pérez, John C.

2012-01-01

Interactions with exposed subendothelial extracellular proteins and cellular integrins (endothelial cells, platelets and lymphocytes) can cause alterations in the hemostatic system associated with atherothrombotic processes. Many molecules found in snake venoms induce pathophysiological changes in humans, cause edema, hemorrhage, and necrosis. Disintegrins are low molecular weight, non-enzymatic proteins found in snake venom that mediate changes by binding to integrins of platelets or other cells and prevent binding of the natural ligands such as fibrinogen, fibronectin or vitronectin. Disintegrins are of great biomedical importance due to their binding affinities resulting in the inhibition of platelet aggregation, adhesion of cancer cells, and induction of signal transduction pathways. RT-PCR was used to obtain a 216 bp disintegrin cDNA from a C. s. scutulatus snake venom gland. The cloned recombinant disintegrin called r-mojastin 1 codes for 71 amino acids, including 12 cysteines, and an RGD binding motif. r-Mojastin 1 inhibited platelet adhesion to fibronectin with an IC50 of 58.3 nM and ADP-induced platelet aggregation in whole blood with an IC50 of 46 nM. r-Mojastin 1 was also tested for its ability to inhibit platelet ATP release using PRP resulting with an IC50 of 95.6 nM. MALDI-TOF mass spectrum analysis showed that r-mojastin has a mass of 7.9509 kDa. PMID:20598348

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

PubMed Central

García, Andrés J.; Vega, María D.; Boettiger, David

1999-01-01

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound α5 and β1 integrin subunits but not αv or β3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of α5β1 integrin bound to Fn, and differentiation was inhibited by anti-α5, but not anti-αv, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications. PMID:10069818

Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

NASA Astrophysics Data System (ADS)

Vogel, Sarah; Arnoldini, Simon; Möller, Stephanie; Schnabelrauch, Matthias; Hempel, Ute

2016-11-01

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

Effect of the Direct Renin Inhibitor Aliskiren on Urinary Albumin Excretion in Spontaneous Type 2 Diabetic KK-A y Mouse

PubMed Central

Furukawa, Masako; Horikoshi, Satoshi; Funabiki, Kazuhiko; Tomino, Yasuhiko

2013-01-01

Objective. Although angiotensin II-mediated inflammation and extracellular matrix accumulation are considered to be associated with the progression of diabetic nephropathy, these processes have not yet been sufficiently clarified. The objective of this study was to determine whether the correction of the abnormal renal expression of MMPs and its inhibitors (MMPs/TIMPs) and cytokines following the administration of aliskiren to KK-A y mice results in a renoprotective effect. Methods. KK-A y mice were divided into two groups, that is, untreated (saline) and treated (aliskiren) groups. Systolic BP, HbA1c levels, and the albumin-creatinine ratio (ACR) were measured. The renal expression of MMPs/TIMPs, fibronectin, type IV collagen, MCP-1, and (pro)renin receptor ((P)RR) was examined using real-time PCR and/or immunohistochemical staining. Renal MAPK and NF-κB activity were also examined by Western blot analyses and ELISA, respectively. Results. Significant decreases in systolic BP and ACR levels were observed in treated KK-A y mice compared with the findings in untreated KK-A y mice. Furthermore, increases in MMPs/TIMPs, fibronectin, type IV collagen, MCP-1, and (P)RR expression, in addition to MAPK and NF-κB activity, were significantly attenuated by aliskiren administration. Conclusions. It appears that aliskiren improves albuminuria and renal fibrosis by regulating inflammation and the alteration of collagen synthesis and degradation. PMID:23819050

Quantitative analysis of collagens and fibronectin expression in human right ventricular hypertrophy.

PubMed

Peters, T H; Sharma, H S; Yilmaz, E; Bogers, A J

1999-06-30

One of the main features in human tetralogy of Fallot (TF) is right ventricular hypertrophy (RVH) due to pressure (sub-pulmonary stenosis) and volume overload (ventricular septal defect). Currently, primary correction at a young age is the treatment of choice. To unravel the role of extracellular matrix in RVH, we examined myocardial expression of collagens and fibronectin in TF patients with primary correction (TF1, age 0.7 +/- 0.2 yr.), secondary surgery (TF2, age 36.9 +/- 4.6 yr), and in age-matched control patients. Sirius red staining quantified by video imaging showed significantly increased interstitial staining for collagens in both TF1 and TF2 groups as compared to respective controls. Fibronectin was expressed in extracellular spaces, perivascular regions, and in some cardiomyocytes. Quantitative analysis of fibronectin revealed increased expression in only TF1 group as compared to respective control. Our results indicate an increased amount of myocardial extracellular matrix deposition as a sign of fibrosis during RVH in patients with TF.

klf2a couples mechanotransduction and zebrafish valve morphogenesis through fibronectin synthesis

PubMed Central

Steed, Emily; Faggianelli, Nathalie; Roth, Stéphane; Ramspacher, Caroline; Concordet, Jean-Paul; Vermot, Julien

2016-01-01

The heartbeat and blood flow signal to endocardial cell progenitors through mechanosensitive proteins that modulate the genetic program controlling heart valve morphogenesis. To date, the mechanism by which mechanical forces coordinate tissue morphogenesis is poorly understood. Here we use high-resolution imaging to uncover the coordinated cell behaviours leading to heart valve formation. We find that heart valves originate from progenitors located in the ventricle and atrium that generate the valve leaflets through a coordinated set of endocardial tissue movements. Gene profiling analyses and live imaging reveal that this reorganization is dependent on extracellular matrix proteins, in particular on the expression of fibronectin1b. We show that blood flow and klf2a, a major endocardial flow-responsive gene, control these cell behaviours and fibronectin1b synthesis. Our results uncover a unique multicellular layering process leading to leaflet formation and demonstrate that endocardial mechanotransduction and valve morphogenesis are coupled via cellular rearrangements mediated by fibronectin synthesis. PMID:27221222

Fibronectin is an acute phase reactant in mice.

PubMed

Dyck, R F; Rogers, S L

1985-01-01

Tissue injury and inflammation are potent stimuli for the immediate increased synthesis of several plasma proteins collectively known as acute phase phase reactants. This dramatic phenomenon is thought to play an important role in inflammation and tissue repair. Plasma fibronectin is a normal plasma glycoprotein and a major non-specific opsonin apparently involved in maintaining the integrity of the mononuclear phagocytic system. Because of its ability to mediate clearance of intravascular particulate matter, increased production following tissue injury could be of benefit to the organism. We now report that plasma fibronectin is a significant acute phase reactant in mice with levels increasing from a baseline mean value of 257 ug/ml to 595 ug/ml by 24 hours (p less than 0.01) after a subcutaneous injection of silver nitrate. Similar findings were observed when subcutaneous casein was used as the acute phase stimulus. This data provides further circumstantial evidence that plasma fibronectin is involved in host defence and tissue repair.

Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

PubMed Central

Brunner, Molly; Millon-Frémillon, Angélique; Chevalier, Genevieve; Nakchbandi, Inaam A.; Mosher, Deane; Block, Marc R.

2011-01-01

The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization. PMID:21768292

Optimization strategies for electrospun silk fibroin tissue engineering scaffolds

PubMed Central

Meinel, Anne J.; Kubow, Kristopher E.; Klotzsch, Enrico; Garcia-Fuentes, Marcos; Smith, Michael L.; Vogel, Viola; Merkle, Hans P.; Meinel, Lorenz

2013-01-01

As a contribution to the functionality of scaffolds in tissue engineering, here we report on advanced scaffold design through introduction and evaluation of topographical, mechanical and chemical cues. For scaffolding, we used silk fibroin (SF), a well established biomaterial. Biomimetic alignment of fibers was achieved as a function of the rotational speed of the cylindrical target during electrospinning of a SF solution blended with polyethylene oxide. Seeding fibrous SF scaffolds with human mesenchymal stem cells (hMSC) demonstrated that fiber alignment could guide hMSC morphology and orientation demonstrating the impact of scaffold topography on the engineering of oriented tissues. Beyond currently established methodologies to measure bulk properties, we assessed the mechanical properties of the fibers by conducting extension at breakage experiments on the level of single fibers. Chemical modification of the scaffolds was tested using donor/acceptor fluorophore labeled fibronectin. Fluorescence resonance energy transfer imaging allowed to assess the conformation of fibronectin when adsorbed on the SF scaffolds, and demonstrated an intermediate extension level of its subunits. Biological assays based on hMSC showed enhanced cellular adhesion and spreading as a result of fibronectin adsorbed on the scaffolds. Our studies demonstrate the versatility of SF as a biomaterial to engineer modified fibrous scaffolds and underscore the use of biofunctionally relevant analytical assays to optimize fibrous biomaterial scaffolds. PMID:19233463

Cellular Interaction of Integrin α3β1 with Laminin 5 Promotes Gap Junctional Communication

PubMed Central

Lampe, Paul D.; Nguyen, Beth P.; Gil, Susana; Usui, Marcia; Olerud, John; Takada, Yoshikazu; Carter, William G.

1998-01-01

Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin α3β1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin α3β1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking α3β1–laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via α3β1 promotes GJIC that integrates individual cells into synchronized epiboles. PMID:9852164

Dynamic contact angle analysis of protein adsorption on polysaccharide multilayer's films for biomaterial reendothelialization.

PubMed

Benni, Safiya; Avramoglou, Thierry; Hlawaty, Hanna; Mora, Laurence

2014-01-01

Atherosclerosis is a major cardiovascular disease. One of the side effects is restenosis. The aim of this work was to study the coating of stents by dextran derivates based polyelectrolyte's multilayer (PEM) films in order to increase endothelialization of injured arterial wall after stent implantation. Films were composed with diethylaminoethyl dextran (DEAE) as polycation and dextran sulphate (DS) as polyanion. One film was composed with 4 bilayers of (DEAE-DS)4 and was labeled D-. The other film was the same as D- but with an added terminal layer of DEAE polycation: (DEAE-DS)4-DEAE (labeled D+). The dynamic adsorption/desorption of proteins on the films were characterized by dynamic contact angle (DCA) and atomic force microscopy (AFM). Human endothelial cell (HUVEC) adhesion and proliferation were quantified and correlated to protein adsorption analyzed by DCA for fibronectin, vitronectin, and bovine serum albumin (BSA). Our results showed that the endothelial cell response was optimal for films composed of DS as external layer. Fibronectin was found to be the only protein to exhibit a reversible change in conformation after desorption test. This behavior was only observed for (DEAE-DS)4 films. (DEAE-DS)4 films could enhance HUVEC proliferation in agreement with fibronectin ability to easily change from conformation.

Dynamic Contact Angle Analysis of Protein Adsorption on Polysaccharide Multilayer's Films for Biomaterial Reendothelialization

PubMed Central

Benni, Safiya; Mora, Laurence

2014-01-01

Atherosclerosis is a major cardiovascular disease. One of the side effects is restenosis. The aim of this work was to study the coating of stents by dextran derivates based polyelectrolyte's multilayer (PEM) films in order to increase endothelialization of injured arterial wall after stent implantation. Films were composed with diethylaminoethyl dextran (DEAE) as polycation and dextran sulphate (DS) as polyanion. One film was composed with 4 bilayers of (DEAE-DS)4 and was labeled D−. The other film was the same as D− but with an added terminal layer of DEAE polycation: (DEAE-DS)4-DEAE (labeled D+). The dynamic adsorption/desorption of proteins on the films were characterized by dynamic contact angle (DCA) and atomic force microscopy (AFM). Human endothelial cell (HUVEC) adhesion and proliferation were quantified and correlated to protein adsorption analyzed by DCA for fibronectin, vitronectin, and bovine serum albumin (BSA). Our results showed that the endothelial cell response was optimal for films composed of DS as external layer. Fibronectin was found to be the only protein to exhibit a reversible change in conformation after desorption test. This behavior was only observed for (DEAE-DS)4 films. (DEAE-DS)4 films could enhance HUVEC proliferation in agreement with fibronectin ability to easily change from conformation. PMID:25276808

Alteration of the Helicobacter pylori membrane proteome in response to changes in environmental salt concentration

PubMed Central

Voss, Bradley J.; Loh, John T.; Hill, Salisha; Rose, Kristie L.; McDonald, W. Hayes; Cover, Timothy L.

2015-01-01

Purpose Helicobacter pylori infection and a high dietary salt intake are each risk factors for the development of gastric cancer. We hypothesize that changes in environmental salt concentrations lead to alterations in the H. pylori membrane proteome. Experimental Design Label-free and iTRAQ methods were used to identify H. pylori proteins that change in abundance in response to alterations in environmental salt concentrations. In addition, we biotinylated intact bacteria that were grown under high- or low-salt conditions, and thereby analyzed salt-induced changes in the abundance of surface-exposed proteins. Results Proteins with increased abundance in response to high salt conditions included CagA, the outer membrane protein HopQ, and fibronectin domain-containing protein HP0746. Proteins with increased abundance in response to low salt conditions included VacA, two VacA-like proteins (ImaA and FaaA), outer-membrane iron transporter FecA3, and several proteins involved in flagellar activity. Consistent with the proteomic data, bacteria grown in high salt conditions exhibited decreased motility compared to bacteria grown in lower salt conditions. Conclusions and clinical relevance Alterations in the H. pylori membrane proteome in response to high salt conditions may contribute to the increased risk of gastric cancer associated with a high salt diet. PMID:26109032

Cellular Interactions and Immune Response of Spherical Nucleic Acid (SNA) Nanoconjugates

NASA Astrophysics Data System (ADS)

Massich, Matthew David

Spherical nucleic acid (SNA) nanoconjugates consist of a densely packed monolayer shell of highly-oriented oligonucleotides covalently bound to a gold nanoparticle core. The nanoconjugates exhibit several important qualities, which make them useful for various biological applications, such as antisense gene regulation strategies and the intracellular detection of biomolecules. The focus of this thesis was to characterize the nanoconjugates interaction with cultured cells and specifically the immune response to their intracellular presence. The immune response of macrophage cells to internalized nanoconjugates was studied, and due to the dense functionalization of oligonucleotides on the surface of the nanoparticle and the resulting high localized salt concentration the innate immune response to the nanoconjugates is ˜25-fold less when compared to a lipoplex carrying the same sequence. Additionally, genome-wide expression profiling was used to study the biological response of cultured cells to the nanoconjugates. The biological response of HeLa cells to gold nanoparticles stabilized by weakly bound ligands was significant, yet when these same nanoparticles were stably functionalized with covalently attached oligonucleotides the cells showed no measurable response. In human keratinocytes, the oligonucleotide sequences caused 427 genes to be differentially expressed when complexed with Dharmafect, but when the oligonucleotides were conjugated to nanoparticles only 7 genes were differentially expressed. Beyond characterizing the cellular interactions and immune response of the nanoconjugates, the optimal length of siRNA (from 19--34 base pairs) that induces the most gene knockdown while maintaining limited immune activation was determined to be 24 base pairs. Further, the SNAs were shown to be useful as a potential antiviral gene therapy by demonstrating approximately 50% knockdown of the Ebola VP35 gene. Lastly, a scanning probe-enabled method was used to rapidly create nanoscale fibronectin patterns over large areas with a range of feature sizes, thereby opening the field of nanocombinatorics. This allowed the investigation of the relationship between fibronectin feature size and stem cell fate. MSCs cultured on nanoscale fibronectin features directed differentiation toward osteogenesis to a greater extent than cells grown on both microscale features and cells grown on non-patterned fibronectin substrates with osteogenic inducing media, demonstrating a new method for controlling stem cell fate.

The Structures of Fibronectin Adsorbed on Polyelectrolyte Thin Films

NASA Astrophysics Data System (ADS)

Shin, Kwanwoo; Satija, Sushil; Fang, Xiao-Hua; Li, Bin-Quan; Nadine, Pernodet; Miriam, Rafailovich; Sokolov, Jonathan; Arach, Goldar; Roser, Steve

2002-03-01

We have shown that it is possible to form a fibrilar network of fibronectin on a polyelectrolyte polymer film whose dimensions are similar to those reported on the extra cellular matrix. The fibronectin network was observed to form only when the charge density of the polymer was in excess of the natural charge density of the cell wall. Furthermore, the self-organized fibronectin layer was much thicker than the polymer film, indicating that long ranged interaction may play a key role in the assembly process. It is therefore important to understand the structure of the polymer layer/protein interface. Here we report on a neutron reflectivity study where we explore the structure of the polyelectrolyte layer, in this case sulfonated polystyrene (PSS_x.), with varying degree of sulfonation (x<30%), as a function of sulfur content and counter ion concentration. These results are then correlated with systemic study of the adsorption and the multilayer formation of fibronectin as a function of incubation time for various sulfonation levels of PSS_x. Furthermore, the surface charge on the substrates can be strongly influenced by the presence of salt ions, it is important to understand changes due to electrostatic interactions occurring in the various salt conditions. Complementary X-ray reflection was used to determine the salt density profile associating with the internal ionic polymer matrix. This work was funded in part of the NSF-MRSEC program.

Interaction of intraocular lenses with fibronectin and human lens epithelial cells: Effect of chemical composition and aging.

PubMed

Tortolano, Lionel; Serrano, Carole; Jubeli, Emile; Saunier, Johanna; Yagoubi, Najet

2015-12-01

The aim of this study is to investigate in vitro interactions between hydrophobic acrylate intraocular lenses (IOLs) and their biological environment. The influence of lens chemical composition and aging on fibronectin (FN) adsorption and on IOLs cytotoxicity on human lens epithelial cells was examined. Cytotoxicity of acrylate monomers used in IOLs manufacture was also investigated. Four different IOLs were included in the study: Acrysof(®), Tecnis(®), EnVista(®), and iSert(®). Implants were artificially aged in a xenon arc chamber to simulate 2 years of light exposure. Fibronectin adsorption on IOL surface was quantified using ELISA and correlated to surface roughness determined with AFM. Direct contact cytotoxicity was determined with the MTT assay and cell morphology was observed with light microscopy. Results showed that fibronectin adsorption did not differ significantly among IOLs, whatever their chemical composition. Moreover, aging conditions did not impact fibronectin adsorption. All IOLs were biocompatible even after applying 2-year aging conditions, with cell viability higher than 70%. Five acrylate monomers appeared to be toxic in the range of concentrations tested, but no monomer release from the IOLs could be detected during accelerated 2-year incubation with saline solution. This study did not reveal an influence of chemical composition and aging on protein adsorption and on biocompatibility. © 2015 Wiley Periodicals, Inc.

Fibronectin EDA forms the chronic fibrotic scar after contusive spinal cord injury.

PubMed

Cooper, John G; Jeong, Su Ji; McGuire, Tammy L; Sharma, Sripadh; Wang, Wenxia; Bhattacharyya, Swati; Varga, John; Kessler, John A

2018-04-27

Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is an impediment to axonal regeneration. Fibrotic scarring is characterized by the accumulation of fibronectin, collagen, and fibroblasts at the lesion site. The mechanisms regulating fibrotic scarring after SCI and its effects on axonal elongation and functional recovery are not well understood. In this study, we examined the effects of eliminating an isoform of fibronectin containing the Extra Domain A domain (FnEDA) on both fibrosis and on functional recovery after contusion SCI using male and female FnEDA-null mice. Eliminating FnEDA did not reduce the acute fibrotic response but markedly diminished chronic fibrotic scarring after SCI. Glial scarring was unchanged after SCI in FnEDA-null mice. We found that FnEDA was important for the long-term stability of the assembled fibronectin matrix during both the subacute and chronic phases of SCI. Motor functional recovery was significantly improved, and there were increased numbers of axons in the lesion site compared to wildtype mice, suggesting that the chronic fibrotic response is detrimental to recovery. Our data provide insight into the mechanisms of fibrosis after SCI and suggest that disruption of fibronectin matrix stability by targeting FnEDA represents a potential therapeutic strategy for promoting recovery after SCI. Copyright © 2018 Elsevier Inc. All rights reserved.

Glioma cell dispersion is driven by α5 integrin-mediated cell-matrix and cell-cell interactions.

PubMed

Blandin, Anne-Florence; Noulet, Fanny; Renner, Guillaume; Mercier, Marie-Cécile; Choulier, Laurence; Vauchelles, Romain; Ronde, Philippe; Carreiras, Franck; Etienne-Selloum, Nelly; Vereb, Gyorgy; Lelong-Rebel, Isabelle; Martin, Sophie; Dontenwill, Monique; Lehmann, Maxime

2016-07-01

Glioblastoma multiform (GBM) is the most common and most aggressive primary brain tumor. The fibronectin receptor, α5 integrin is a pertinent novel therapeutic target. Despite numerous data showing that α5 integrin support tumor cell migration and invasion, it has been reported that α5 integrin can also limit cell dispersion by increasing cell-cell interaction. In this study, we showed that α5 integrin was involved in cell-cell interaction and gliomasphere formation. α5-mediated cell-cell cohesion limited cell dispersion from spheroids in fibronectin-poor microenvironment. However, in fibronectin-rich microenvironment, α5 integrin promoted cell dispersion. Ligand-occupied α5 integrin and fibronectin were distributed in fibril-like pattern at cell-cell junction of evading cells, forming cell-cell fibrillar adhesions. Activated focal adhesion kinase was not present in these adhesions but was progressively relocalized with α5 integrin as cell migrates away from the spheroids. α5 integrin function in GBM appears to be more complex than previously suspected. As GBM overexpressed fibronectin, it is most likely that in vivo, α5-mediated dissemination from the tumor mass overrides α5-mediated tumor cell cohesion. In this respect, α5-integrin antagonists may be useful to limit GBM invasion in brain parenchyma. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Substrate engagement of integrins α5β1 and αvβ3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

PubMed Central

Missirlis, Dimitris; Haraszti, Tamás; Scheele, Catharina v. C.; Wiegand, Tina; Diaz, Carolina; Neubauer, Stefanie; Rechenmacher, Florian; Kessler, Horst; Spatz, Joachim P.

2016-01-01

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5β1 and αvβ3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αvβ3 or α5β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration. PMID:26987342

N-cadherin adhesive interactions modulate matrix mechanosensing and fate commitment of mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Cosgrove, Brian D.; Mui, Keeley L.; Driscoll, Tristan P.; Caliari, Steven R.; Mehta, Kush D.; Assoian, Richard K.; Burdick, Jason A.; Mauck, Robert L.

2016-12-01

During mesenchymal development, the microenvironment gradually transitions from one that is rich in cell-cell interactions to one that is dominated by cell-ECM (extracellular matrix) interactions. Because these cues cannot readily be decoupled in vitro or in vivo, how they converge to regulate mesenchymal stem cell (MSC) mechanosensing is not fully understood. Here, we show that a hyaluronic acid hydrogel system enables, across a physiological range of ECM stiffness, the independent co-presentation of the HAVDI adhesive motif from the EC1 domain of N-cadherin and the RGD adhesive motif from fibronectin. Decoupled presentation of these cues revealed that HAVDI ligation (at constant RGD ligation) reduced the contractile state and thereby nuclear YAP/TAZ localization in MSCs, resulting in altered interpretation of ECM stiffness and subsequent changes in downstream cell proliferation and differentiation. Our findings reveal that, in an evolving developmental context, HAVDI/N-cadherin interactions can alter stem cell perception of the stiffening extracellular microenvironment.

EFFECT OF GROWTH FACTOR-FIBRONECTIN MATRIX INTERACTION ON RAT TYPE II CELL ADHESION AND DNA SYTHESIS

EPA Science Inventory

ABSTRACT

Type II cells attach, migrate and proliferate on a provisional fibronectin-rich matrix during alveolar wall repair after lung injury. The combination of cell-substratum interactions via integrin receptors and exposure to local growth factors are likely to initiat...

Recombinant fibronectin fragment III8-10/polylactic acid hybrid nanofibers enhance the bioactivity of titanium surface.

PubMed

Guillem-Marti, Jordi; Boix-Lemonche, Gerard; Gugutkov, Dencho; Ginebra, Maria-Pau; Altankov, George; Manero, Jose M

2018-04-01

To develop a nanofiber (NF)-based biomimetic coating on titanium (Ti) that mimics the complex spatiotemporal organization of the extracellular matrix (ECM). Recombinant cell attachment site (CAS) of fibronectin type III8-10 domain was co-electrospun with polylactic acid (PLA) and covalently bound on polished Ti discs. Osteoblast-like SaOS-2 cells were used to evaluate their complex bioactivity. A significant increase of cell spreading was found on CAS/PLA hybrid NFs, followed by control pure PLA NFs and bare Ti discs. Cell proliferation showed similar trend being about twice higher on CAS/PLA NFs. The significantly increased ALP activity at day 21 indicated an enhanced differentiation of SaOS-2 cells. Coating of Ti implants with hybrid CAS/PLA NFs may improve significantly their osseointegration potential.

Mechanism of mast cell adhesion to human tenocytes in vitro.

PubMed

Behzad, Hayedeh; Tsai, Shu-Huei; Nassab, Paulina; Mousavizadeh, Rouhollah; McCormack, Robert G; Scott, Alex

2015-01-01

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Structural and functional analysis of an anchorless fibronectin-binding protein FBPS from Gram-positive bacterium Streptococcus suis.

PubMed

Musyoki, Abednego Moki; Shi, Zhongyu; Xuan, Chunling; Lu, Guangwen; Qi, Jianxun; Gao, Feng; Zheng, Beiwen; Zhang, Qiangmin; Li, Yan; Haywood, Joel; Liu, Cuihua; Yan, Jinghua; Shi, Yi; Gao, George F

2016-11-29

The anchorless fibronectin-binding proteins (FnBPs) are a group of important virulence factors for which the structures are not available and the functions are not well defined. In this study we performed comprehensive studies on a prototypic member of this group: the fibronectin-/fibrinogen-binding protein from Streptococcus suis (FBPS). The structures of the N- and C-terminal halves (FBPS-N and FBPS-C), which together cover the full-length protein in sequence, were solved at a resolution of 2.1 and 2.6 Å, respectively, and each was found to be composed of two domains with unique folds. Furthermore, we have elucidated the organization of these domains by small-angle X-ray scattering. We further showed that the fibronectin-binding site is located in FBPS-C and that FBPS promotes the adherence of S suis to host cells by attaching the bacteria via FBPS-N. Finally, we demonstrated that FBPS functions both as an adhesin, promoting S suis attachment to host cells, and as a bacterial factor, activating signaling pathways via β1 integrin receptors to induce chemokine production.

MicroRNA MiR-17 retards tissue growth and represses fibronectin expression.

PubMed

Shan, Sze Wan; Lee, Daniel Y; Deng, Zhaoqun; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W; Zhang, Yaou; Xuan, Jim W; Yee, Siu-Pok; Siragam, Vinayakumar; Yang, Burton B

2009-08-01

MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-17~92 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We anticipate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.

The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

PubMed

Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

2014-01-01

Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile. Copyright © 2013. Published by Elsevier GmbH.

CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and Fibronectin-enriched matrices

PubMed Central

McFarlane, Suzanne; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; Waugh, David J.J.

2015-01-01

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche. PMID:26447611

Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsieh, Pei-Fang; Graduate Institute of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, Taiwan; Liu, Shu-Fen

Background: Mitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. Materials and methods: In vitro fibrosis model was established by treatment with transforming growth factor-β1 (TGF-β1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis andmore » fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). Results: Under renal fibrosis conditions, TGF-β1 (5 ng/ml) increased ROS. Simultaneously, TGF-β1-induced extracellular fibronectin by ELISA assay. In addition, TGF-β1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2 μg/ml) expression vector dramatically reverse TGF-β1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2 μg/ml) significantly increased the expression of fibronectin of both intercellular and extracellular origins in NRK-49F cells. Discussion: These finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells.« less

Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis.

PubMed

Hsieh, Pei-Fang; Liu, Shu-Fen; Hung, Tsung-Jen; Hung, Chien-Ya; Liu, Guo-Zheng; Chuang, Lea-Yea; Chen, Mei-Fen; Wang, Jue-Long; Shi, Ming-Der; Hsu, Chen Hung; Shiue, Yow-Ling; Yang, Yu-Lin

2016-11-15

Mitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. In vitro fibrosis model was established by treatment with transforming growth factor-β1 (TGF-β1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis and fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). Under renal fibrosis conditions, TGF-β1 (5ng/ml) increased ROS. Simultaneously, TGF-β1-induced extracellular fibronectin by ELISA assay. In addition, TGF-β1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2μg/ml) expression vector dramatically reverse TGF-β1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2μg/ml) significantly increased the expression of fibronectin of both intercellular and extracellular origins in NRK-49F cells. These finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Nickel-induced Epithelial-Mesenchymal Transition by Reactive Oxygen Species Generation and E-cadherin Promoter Hypermethylation*

PubMed Central

Wu, Chih-Hsien; Tang, Sheau-Chung; Wang, Po-Hui; Lee, Huei; Ko, Jiunn-Liang

2012-01-01

Epithelial-mesenchymal transition (EMT) is considered a critical event in the pathogenesis of lung fibrosis and tumor metastasis. During EMT, the expression of differentiation markers switches from cell-cell junction proteins such as E-cadherin to mesenchymal markers such as fibronectin. Although nickel-containing compounds have been shown to be associated with lung carcinogenesis, the role of nickel in the EMT process in bronchial epithelial cells is not clear. The aim of this study was to examine whether nickel contributes to EMT in human bronchial epithelial cells. We also attempted to clarify the mechanisms involved in NiCl2-induced EMT. Our results showed that NiCl2 induced EMT phenotype marker alterations such as up-regulation of fibronectin and down-regulation of E-cadherin. In addition, the potent antioxidant N-acetylcysteine blocked EMT and expression of HIF-1α induced by NiCl2, whereas the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine restored the down-regulation of E-cadherin induced by NiCl2. Promoter hypermethylation of E-cadherin, determined by quantitative real time methyl-specific PCR and bisulfate sequencing, was also induced by NiCl2. These results shed new light on the contribution of NiCl2 to carcinogenesis. Specifically, NiCl2 induces down-regulation of E-cadherin by reactive oxygen species generation and promoter hypermethylation. This study demonstrates for the first time that nickel induces EMT in bronchial epithelial cells. PMID:22648416

Role of plasma fibronectin in the foreign body response to biomaterials.

PubMed

Keselowsky, Benjamin G; Bridges, Amanda W; Burns, Kellie L; Tate, Ciara C; Babensee, Julia E; LaPlaca, Michelle C; García, Andrés J

2007-09-01

Host responses to biomaterials control the biological performance of implanted medical devices. Upon implantation, synthetic materials adsorb biomolecules, which trigger an inflammatory cascade comprising coagulation, leukocyte recruitment/adhesion, and foreign body reaction. The foreign body reaction and ensuing fibrous encapsulation severely limit the in vivo performance of numerous biomedical devices. While it is well established that plasma fibrinogen and secreted cytokines modulate leukocyte recruitment and maturation into foreign body giant cells, mediators of chronic inflammation and fibrous encapsulation of implanted biomaterials remain poorly understood. Using plasma fibronectin (pFN) conditional knock-out mice, we demonstrate that pFN modulates the foreign body response to polyethylene terephthalate disks implanted subcutaneously. Fibrous collagenous capsules were two-fold thicker in mice depleted of pFN compared to controls. In contrast, deletion of pFN did not alter acute leukocyte recruitment to the biomaterial, indicating that pFN modulates chronic fibrotic responses. The number of foreign body giant cells associated with the implant was three times higher in the absence of pFN while macrophage numbers were not different, suggesting that pFN regulates the formation of biomaterial-associated foreign body giant cells. Interestingly, cellular FN (cFN) was present in the capsules of both normal and pFN-depleted mice, suggesting that cFN could not compensate for the loss of pFN. These results implicate pFN in the host response to implanted materials and identify a potential target for therapeutic intervention to enhance the biological performance of biomedical devices.

Leiomyoma Cells in 3-Dimensional Cultures Demonstrate an Attenuated Response to Fasudil, a Rho-Kinase Inhibitor, When Compared to 2-Dimensional Cultures

PubMed Central

Malik, Minnie; Britten, Joy; Segars, James

2014-01-01

Uterine leiomyomata are common benign tumors in women of reproductive age and demonstrate an attenuated response to mechanical signaling that involves Rho and integrins. To further characterize the impairment in Rho signaling, we studied the effect of Rho-kinase inhibitor, fasudil, on extracellular matrix production, in 2-dimensional (2D) and 3-dimensional (3D) cultures of leiomyoma and myometrial cells. Leiomyoma 2D cultures demonstrated a rapid decrease in gene transcripts and protein for fibronectin, procollagen 1A, and versican. In 3D cultures, fibronectin and procollagen 1A proteins demonstrated increased levels at lower concentrations of fasudil, followed by a concentration-dependent decrease. Versican protein increased up to 3-fold, whereas fibromodulin demonstrated a significant decrease of 1.92-fold. Myometrial 2D or 3D cultures demonstrated a decrease in all proteins after 72 hours of treatment. The 3D leiomyoma cultures demonstrated a significant increase in active RhoA, followed by a concentration-dependent decrease at higher concentrations. A concentration-dependent increase in phospho-extracellular regulated signal kinase and proapoptotic protein Bax was observed in 3D leiomyoma cultures. Fasudil relaxed the contraction of the 3D collagen gels caused by myometrium and leiomyoma cell growth. These findings indicate that the altered state of Rho signaling in leiomyoma was more clearly observed in 3D cultures. The results also suggest that fasudil may have clinical applicability for treatment of uterine leiomyoma. PMID:25084783

Effect of antibiotic exposure on Nugent score among pregnant women with and without bacterial vaginosis.

PubMed

Anderson, Brenna; Zhao, Yuan; Andrews, William W; Dudley, Donald J; Sibai, Baha; Iams, Jay D; Wapner, Ronald J; Varner, Michael W; Caritis, Steve N; O'Sullivan, Mary Jo

2011-04-01

To evaluate whether vaginal flora is altered by antibiotic exposure and associated with a risk of preterm birth, particularly among women with initially normal vaginal flora. This was a secondary analysis of a randomized trial of metronidazole and erythromycin for the prevention of preterm birth among women with a positive fetal fibronectin test. Vaginal swabs for Nugent Gram stain score were collected for classification of bacterial vaginosis before and after antibiotic exposure and read at a central laboratory. Change in Nugent score was assessed for women with (score 7 or higher) or without (score lower than 7) bacterial vaginosis. Linear regression analysis evaluated whether change in Nugent score was associated with preterm birth. Two hundred women without and 69 women with bacterial vaginosis had Gram stain performed before and after antibiotic therapy. Median Nugent score for all women declined from 4.0 to 2.0 after antibiotic therapy (P<.001). Nugent score declined both for those without (from 2.0 to 1.5, P=.11) and, more dramatically, those with bacterial vaginosis (from 8.0 to 3.0, P<.01). The components of the Nugent score that were affected by antibiotic exposure were similar among women with and without bacterial vaginosis. Antibiotic exposure and the change in Nugent score were unrelated to preterm birth among bacterial vaginosis-negative women. Antibiotic exposure is not associated with preterm birth and does not worsen Nugent score among women with normal vaginal flora and positive fetal fibronectin. II.

Progression of renal fibrosis in congenital CKD model rats with reduced number of nephrons.

PubMed

Yasuda, Hidenori; Tochigi, Yuki; Katayama, Kentaro; Suzuki, Hiroetsu

2017-06-14

A congenital reduction in the number of nephrons is a critical risk factor for both onset of chronic kidney disease (CKD) and its progression to end-stage kidney disease (ESKD). Hypoplastic kidney (HPK) rats have only about 20% of the normal number of nephrons and show progressive CKD. This study used an immunohistological method to assess glomerular and interstitial pathogenesis in male HPK rats aged 35-210days. CD68 positive-macrophages were found to infiltrate into glomeruli in HPK rats aged 35 and 70days and to infiltrate into interstitial tissue in rats aged 140 and 210days. HPK rats aged 35 and 70days showed glomerular hypertrophy, loss of normal linear immunostaining of podocine, and increased expression of PDGFr-β, TGF-β, collagens, and fibronectin, with all of these alterations gradually deteriorating with age. α-SMA-positive myofibroblasts were rarely detected in glomerular tufts, whereas α-SMA-positive glomerular parietal epithelium (GPE) cells were frequently observed along Bowman's capsular walls. The numbers of PDGFr-β-positive fibroblasts in interstitial tissue were increased in rats aged 35days and older, whereas interstitial fibrosis, characterized by the increased expression of tubular PDGF-BB, the appearance of myofibroblasts doubly positive for PDGFr-β and α-SMA, and increased expression of collagens and fibronectin, were observed in rats aged 70 and older. These results clearly indicate that congenital CKD with only 20% of nephrons cause renal fibrosis in rats. Copyright © 2017 Elsevier GmbH. All rights reserved.

Correlation Between Extravasation and Alterations of Cerebrovascular Laminin and β-Dystroglycan Immunoreactivity Following Cryogenic Lesions in Rats.

PubMed

Kálmán, Mihály; Tóth, László; Szöllosi, Dávid; Oszwald, Erzsébet; Mahalek, Judit; Sadeghian, Sam

2017-11-01

The blood-brain barrier becomes "leaky" following lesions. Former studies revealed that following lesions the immunoreactivity of cerebrovascular laminin becomes detectable whereas that of β-dystroglycan disappears. These alterations may be indicators of glio-vascular decoupling that may result in the impairment of the blood-brain-barrier. This study investigates correlation between the post-lesion extravasation and the above-mentioned immunohistochemical alterations. Following cryogenic lesions, the survival periods lasted 5, 10, 30 minutes, 1 or 12 hours, or 1 day. Some brains were fixed immediately post-lesion. Immunofluorescent reactions were performed in floating sections. The extravasation was detected with immunostaining for plasma fibronectin and rat immunoglobulins. When the survival period was 30 minutes or longer, the area of extravasation corresponded to the area of altered laminin and β-dystroglycan immunoreactivities. Following immediate fixation some laminin immunoreactivity was already detected. The extravasation seemed to precede this early appearance of laminin immunoreactivity. The β-dystroglycan immunoreactivity disappeared later. When the extravasation spread into the corpus callosum, vascular laminin immunoreactivity appeared but the β-dystroglycan immunoreactivity persisted. It seems that extravasation separates the glial and vascular basal laminae, which results in the appearance of laminin immunoreactivity. The disappearance of β-dystroglycan immunoreactivity is neither a condition nor an inevitable consequence of the 2 other phenomena. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

Binding of extracellular matrix proteins to Aspergillus fumigatus conidia.

PubMed Central

Gil, M L; Peñalver, M C; Lopez-Ribot, J L; O'Connor, J E; Martinez, J P

1996-01-01

As detected by confocal immunofluorescence microscopy, binding of fibronectin and laminin appeared to be associated with the protrusions present on the outer cell wall layer of resting Aspergillus fumigatus conidia. Flow cytometry confirmed that binding of laminin to conidia was dose dependent and saturable. Laminin binding was virtually eliminated in trypsin-treated organisms, thus suggesting the protein nature of the binding site. Conidia were also able to specifically adhere to laminin immobilized on microtiter plates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) with laminin and antilaminin antibody of whole conidial homogenates allowed identification, among the complex array of protein and glycoprotein species, of one polypeptide with an apparent molecular mass of 37 kDa which specifically interacts with laminin. The fact that binding of conidia to soluble or immobilized laminin or fibronectin was inhibited by fibronectin or laminin, respectively, suggests the existence of common binding sites for both ligands on the surface of conidia. Intact conidia were also able to adhere to type I and IV collagen immobilized on microtiter plates; adhesion was found to be dose dependent and saturable. Adhesion to immobilized type I and IV collagen was markedly inhibited by laminin and weakly inhibited by fibronectin. Coincubation of conidia with Arg-Gly-Asp (RGD) peptides caused a dose-dependent decrease in binding of cells to immobilized or soluble fibronectin, yet interaction of cells with soluble or immobilized laminin and type I and IV collagen remained unaffected. Interactions described here could be important in mediating attachment of the fungus to host tissues, thus playing a role in the establishment of the disease. PMID:8945572

Behavior of sea urchin primary mesenchyme cells in artificial extracellular matrices.

PubMed

Katow, H

1986-02-01

The primary mesenchyme cells (PMCs) were separated from the mesenchyme blastulae of Pseudocentrotus depressus using differential adhesiveness of these cells to plastic Petri dishes. These cells were incubated in various artificial extracellular matrices (ECMs) including horse serum plasma fibronectin, mouse EHS sarcoma laminin, mouse EHS sarcoma type IV collagen, and porcine skin dermatan sulfate. The cell behavior was monitored by a time-lapse videomicrograph and analysed with a microcomputer. The ultrastructure of the artificial ECM was examined by transmission electron microscopy (TEM), while the ultrastructure of the PMCs was examined by scanning electron microscopy (SEM). The PMCs did not migrate in type IV collagen gel, laminin or dermatan sulfate matrix either with or without collagen gel, whereas PMCs in the matrix which was composed of fibronectin and collagen gel migrated considerably. However, the most active and extensive PMC migration was seen in the matrix which contained dermatan sulfate in addition to fibronectin and collagen gel. This PMC migration involved an increase not only of migration speed but also of proportion of migration-promoted cells. These results support the hypothesis that the mechanism of PMC migration involves fibronectin, collagen and sulfated proteoglycans which contain dermatan sulfate.

Controlled Assembly of Fibronectin Nanofibrils Triggered by Random Copolymer Chemistry.

PubMed

Mnatsakanyan, Hayk; Rico, Patricia; Grigoriou, Eleni; Candelas, Aarón Maturana; Rodrigo-Navarro, Aleixandre; Salmeron-Sanchez, Manuel; Sabater i Serra, Roser

2015-08-19

Fibronectin fibrillogenesis is the physiological process by which cells elaborate a fibrous FN matrix. Poly(ethyl acrylate), PEA, has been described to induce a similar process upon simple adsorption of fibronectin (FN) from a protein solution-in the absence of cells-leading to the so-called material-driven fibronectin fibrillogenesis. Poly(methyl acrylate), PMA, is a polymer with very similar chemistry to PEA, on which FN is adsorbed, keeping the globular conformation of the protein in solution. We have used radical polymerization to synthesize copolymers with controlled EA/MA ratio, seeking to modulate the degree of FN fibrillogenesis. The physicochemical properties of the system were studied using dynamic-mechanical analysis, differential scanning calorimetry, and water contact angle. Both the degree of FN fibrillogenesis and the availability of the integrin binding region of FN directly depend on the percentage of EA in the copolymer, whereas the same total amount of FN was adsorbed regardless the EA/MA ratio. Cell morphology adhesion and differentiation of murine C2C12 were shown to depend on the degree of FN fibrillogenesis previously attained on the material surface. Myogenic differentiation was enhanced on the copolymers with higher EA content, i.e. more interconnected FN fibrils.

The Cellular Form of Human Fibronectin as an Adhesion Target for the S Fimbriae of Meningitis-Associated Escherichia coli

PubMed Central

Sarén, Anne; Virkola, Ritva; Hacker, Jörg; Korhonen, Timo K.

1999-01-01

The adhesion of the S fimbriae of meningitis-associated Escherichia coli O18ac:K1:H7 to the cellular and the plasma forms of human fibronectin was studied. E. coli HB101(pAZZ50) expressing the complete S-fimbria II gene cluster of E. coli O18 adhered to cellular fibronectin (cFn) on glass but not to plasma fibronectin (pFn). Adhesion to cFn was specifically inhibited by neuraminidase treatment of cFn as well as by incubation of the bacteria with sialyl-α2-3-lactose, a receptor analog of the S fimbriae. No significant adhesion to cFn or pFn was detected with E. coli HB101(pAZZ50-67) expressing S fimbriae lacking the SfaS lectin subunit. Strain HB101(pAZZ50) also adhered to a human fibroblast cell culture known to be rich in cFn, and the adhesion was specifically inhibited in the presence of polyclonal antibodies to cFn. The results show that the SfaS lectin of the S fimbriae mediates the adherence of meningitis-associated E. coli to sialyl oligosaccharide chains of cFn. PMID:10225941

Fish Rhabdovirus Cell Entry Is Mediated by Fibronectin

PubMed Central

Bearzotti, Monique; Delmas, Bernard; Lamoureux, Annie; Loustau, Anne-Marie; Chilmonczyk, Stefan; Bremont, Michel

1999-01-01

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family. PMID:10438860

Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells.

PubMed

Bacakova, Marketa; Pajorova, Julia; Stranska, Denisa; Hadraba, Daniel; Lopot, Frantisek; Riedel, Tomas; Brynda, Eduard; Zaloudkova, Margit; Bacakova, Lucie

2017-01-01

Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide- co -glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types.

Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells

PubMed Central

Bacakova, Marketa; Pajorova, Julia; Stranska, Denisa; Hadraba, Daniel; Lopot, Frantisek; Riedel, Tomas; Brynda, Eduard; Zaloudkova, Margit; Bacakova, Lucie

2017-01-01

Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide-co-glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types. PMID:28223803

Establishment of substratum polarity in the blastocoel roof of the Xenopus embryo.

PubMed

Nagel, M; Winklbauer, R

1999-05-01

The fibronectin fibril matrix on the blastocoel roof of the Xenopus gastrula contains guidance cues that determine the direction of mesoderm cell migration. The underlying guidance-related polarity of the blastocoel roof is established in the late blastula under the influence of an instructive signal from the vegetal half of the embryo, in particular from the mesoderm. Formation of an oriented substratum depends on functional activin and FGF signaling pathways in the blastocoel roof. Besides being involved in tissue polarization, activin and FGF also affect fibronectin matrix assembly. Activin treatment of the blastocoel roof inhibits fibril formation, whereas FGF modulates the structure of the fibril network. The presence of intact fibronectin fibrils is permissive for directional mesoderm migration on the blastocoel roof extracellular matrix.

Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

PubMed Central

Shadpour, Hamed; Zawistowski, Jon S.; Herman, Annadele; Hahn, Klaus; Allbritton, Nancy L.

2011-01-01

Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronection coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4 fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties. PMID:21621038

Identification of Novel Pro-Migratory, Cancer-Associated Genes Using Quantitative, Microscopy-Based Screening

PubMed Central

Naffar-Abu-Amara, Suha; Shay, Tal; Galun, Meirav; Cohen, Naomi; Isakoff, Steven J.; Kam, Zvi; Geiger, Benjamin

2008-01-01

Background Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary. Methodology In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features. Conclusions In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion. PMID:18213366

Intravesical Bacillus Calmette-Guérin therapy for murine bladder tumors: initiation of the response by fibronectin-mediated attachment of Bacillus Calmette-Guérin.

PubMed

Ratliff, T L; Palmer, J O; McGarr, J A; Brown, E J

1987-04-01

Intravesical Bacillus Calmette-Guérin (BCG) is considered to be one of the most effective treatments for superficial bladder cancer. Although the mechanisms by which BCG inhibits tumor growth are not known, previous studies have shown that systemic immunization to BCG and the local expression of the immune response in the bladder are associated with a favorable response to BCG therapy. We have investigated the conditions required for the initiation of an immunological response after the intravesical instillation of BCG. Initial histological studies showed that BCG attached to the bladder wall only in areas where the urothelium was damaged by electrocautery and suggested that attachment was associated with the fibrin clot. Quantitative studies verified the histological observations. Minimal BCG attachment (mean less than 10(2) colony forming units) was observed in normal bladders in contrast with a mean of 1.42 X 10(4) colony forming units/bladder in bladders damaged by electrocautery (10 separate experiments). BCG attachment to the bladder wall was durable since organisms were observed in bladders 48 h after instillation. To investigate the proteins to which BCG attached, we tested the binding of BCG to extracellular matrix and inflammatory proteins which comprise a significant portion of the fibrin clot. BCG bound in vitro to coverslips coated in vivo with extracellular matrix proteins but did not bind to control albumin-coated coverslips. BCG also bound to coverslips coated with purified plasma fibronectin but not to coverslips coated with other purified extracellular matrix proteins including laminin, fibrinogen, and type IV collagen. BCG attachment to coverslips coated with either extracellular matrix proteins or purified fibronectin was inhibited by antibodies specific for fibronectin. Moreover, BCG attachment to cauterized bladders in vivo was inhibited by antifibronectin antibodies. These results demonstrate that fibronectin mediates the attachment of BCG to surfaces and suggest that it is the primary component mediating attachment within the bladder. Moreover, the data suggest that the BCG-fibronectin interaction may be a requisite first step for the initiation of the antitumor activity in intravesical BCG for bladder cancer.

Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN α5β1-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION.

PubMed

Mould, A Paul; Askari, Janet A; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A; Humphries, Martin J

2016-09-30

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Extracellular matrix components direct porcine muscle stem cell behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl

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 gelatinmore » 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.« less

Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy.

PubMed Central

Clausell, N.; de Lima, V. C.; Molossi, S.; Liu, P.; Turley, E.; Gotlieb, A. I.; Adelman, A. G.; Rabinovitch, M.

1995-01-01

BACKGROUND--The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury. METHODS--Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility. RESULTS--The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta. CONCLUSIONS--Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury. Images PMID:7626352

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa.

PubMed

Middleton, A M; Chadwick, M V; Nicholson, A G; Dewar, A; Groger, R K; Brown, E J; Wilson, R

2000-10-01

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.

Chymase inhibition prevents fibronectin and myofibrillar loss and improves cardiomyocyte function and LV torsion angle in dogs with isolated mitral regurgitation.

PubMed

Pat, Betty; Chen, Yuanwen; Killingsworth, Cheryl; Gladden, James D; Shi, Ke; Zheng, Junying; Powell, Pamela C; Walcott, Greg; Ahmed, Mustafa I; Gupta, Himanshu; Desai, Ravi; Wei, Chih-Chang; Hase, Naoki; Kobayashi, Tsunefumi; Sabri, Abdelkarim; Granzier, Henk; Denney, Thomas; Tillson, Michael; Dillon, A Ray; Husain, Ahsan; Dell'italia, Louis J

2010-10-12

The left ventricular (LV) dilatation of isolated mitral regurgitation (MR) is associated with an increase in chymase and a decrease in interstitial collagen and extracellular matrix. In addition to profibrotic effects, chymase has significant antifibrotic actions because it activates matrix metalloproteinases and kallikrein and degrades fibronectin. Thus, we hypothesize that chymase inhibitor (CI) will attenuate extracellular matrix loss and LV remodeling in MR. We studied dogs with 4 months of untreated MR (MR; n=9) or MR treated with CI (MR+CI; n=8). Cine MRI demonstrated a >40% increase in LV end-diastolic volume in both groups, consistent with a failure of CI to improve a 25% decrease in interstitial collagen in MR. However, LV cardiomyocyte fractional shortening was decreased in MR versus normal dogs (3.71±0.24% versus 4.81±0.31%; P<0.05) and normalized in MR+CI dogs (4.85±0.44%). MRI with tissue tagging demonstrated an increase in LV torsion angle in MR+CI versus MR dogs. CI normalized the significant decrease in fibronectin and FAK phosphorylation and prevented cardiomyocyte myofibrillar degeneration in MR dogs. In addition, total titin and its stiffer isoform were increased in the LV epicardium and paralleled the changes in fibronectin and FAK phosphorylation in MR+CI dogs. These results suggest that chymase disrupts cell surface-fibronectin connections and FAK phosphorylation that can adversely affect cardiomyocyte myofibrillar structure and function. The greater effect of CI on epicardial versus endocardial titin and noncollagen cell surface proteins may be responsible for the increase in torsion angle in chronic MR.

Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair.

PubMed

Girault, Alban; Chebli, Jasmine; Privé, Anik; Trinh, Nguyen Thu Ngan; Maillé, Emilie; Grygorczyk, Ryszard; Brochiero, Emmanuelle

2015-09-04

Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

Curli mediate bacterial adhesion to fibronectin via tensile multiple bonds

NASA Astrophysics Data System (ADS)

Oh, Yoo Jin; Hubauer-Brenner, Michael; Gruber, Hermann J.; Cui, Yidan; Traxler, Lukas; Siligan, Christine; Park, Sungsu; Hinterdorfer, Peter

2016-09-01

Many enteric bacteria including pathogenic Escherichia coli and Salmonella strains produce curli fibers that bind to host surfaces, leading to bacterial internalization into host cells. By using a nanomechanical force-sensing approach, we obtained real-time information about the distribution of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We found that curliated E. coli and fibronectin formed dense quantized and multiple specific bonds with high tensile strength, resulting in tight bacterial binding. Nanomechanical recognition measurements revealed that approximately 10 bonds were disrupted either sequentially or simultaneously under force load. Thus the curli formation of bacterial surfaces leads to multi-bond structural components of fibrous nature, which may explain the strong mechanical binding of curliated bacteria to host cells and unveil the functions of these proteins in bacterial internalization and invasion.

Modulation of Human Plasma Fibronectin Levels Following Exercise,

DTIC Science & Technology

1988-01-01

forms of this large molecular weight (440 kilodaltons) glycoprotein,(17. While the tissue type is cell-associated and important to cell adhesion and...increased under conditions of pathology, such as in obesity (6). cancer (3). proteinuria (4). diabetic retinopathy (5). and preeclampsia (27). in the absence...Res. 1977: 22:709-716. 27. Stubbs. T.M.. Lazarchick. J.. and Horger. E.O. Plasma fibronectin levels in preeclampsia : A possible biochemical marker

Interaction of Mycobacterium tuberculosis with human respiratory mucosa.

PubMed

Middleton, A M; Chadwick, M V; Nicholson, A G; Dewar, A; Groger, R K; Brown, E J; Ratliff, T L; Wilson, R

2002-01-01

Endobronchial infection is associated with pulmonary tuberculosis in the majority of cases. We have investigated the adherence of Mycobacterium tuberculosis to the human respiratory mucosa. Organ cultures constructed with human tissue were infected with M. tuberculosis in the presence or absence of mycobacterial fibronectin attachment cell surface proteins and examined by scanning electron microscopy. M. tuberculosis adhered mainly to extracellular matrix (ECM) in areas of mucosal damage, but not to ciliated mucosa, intact extruded cells, basement membrane or collagen fibres. Bacteria also adhered to fibrous but not globular mucus and occasionally to healthy unciliated mucosa, open tight junctions and to extruded cells that had degenerated, exposing their contents. There was a significant reduction (p<0.05) in the number of bacteria adhering to ECM after pre-incubation of bacteria with fibronectin and after pre-incubation of the tissue with M. avium fibronectin attachment protein (FAP) and M. bovis antigen 85B protein, in a concentration dependent manner. The combined effect of FAP and antigen 85B protein was significantly greater than either protein alone. Bacterial adherence to fibrous mucus was not influenced by fibronectin. We conclude that M. tuberculosis adheres to ECM in areas of mucosal damage at least in part via FAP and antigen 85B protein.

Is vitronectin the velcro that binds the gametes together?

PubMed

Fusi, F M; Bernocchi, N; Ferrari, A; Bronson, R A

1996-11-01

Evidence has been presented that the adhesion of human spermatozoa to the oolemma is mediated by integrins recognizing the Arg-Gly-Asp sequence (RGD). Fibronectin and vitronectin, glycoproteins that contain functional RGD sequences, are both present on human spermatozoa, and integrins that recognize these ligands have been detected on spermatozoa and eggs. In this work, we studied the effects of oligopeptides specifically designed to block fibronectin or vitronectin receptors on the interaction of human spermatozoa with zona-free hamster oocytes. GRGDdSP, a peptide blocking cell attachment to fibronectin, was without effect, while GdRGDSP, which blocks both fibronectin and vitronectin receptors, significantly inhibited the binding of human spermatozoa to the oolemma of zona-free hamster eggs, in a concentration-dependent manner, over a range 1-100 microM. As these experiments suggested that a vitronectin receptor plays a role in sperm-oolemmal adhesion, we performed a series of experiments studying the effects of exogenous vitronectin, when added to spermatozoa and oocytes, on gamete interactions. Sperm-oolemmal adherence, as well as sperm aggregation, was promoted by vitronectin, over range of 2.2 nM to 1 microM, but only in the presence of calcium ions. We propose that vitronectin released during the sperm acrosome reaction is recognized by both gametes and plays a role in their adhesion.

The eighth fibronectin type III domain of protein tyrosine phosphatase receptor J influences the formation of protein complexes and cell localization.

PubMed

Iuliano, Rodolfo; Raso, Cinzia; Quintiero, Alfina; Pera, Ilaria Le; Pichiorri, Flavia; Palumbo, Tiziana; Palmieri, Dario; Pattarozzi, Alessandra; Florio, Tullio; Viglietto, Giuseppe; Trapasso, Francesco; Croce, Carlo Maria; Fusco, Alfredo

2009-03-01

Regulation of receptor-type phosphatases can involve the formation of higher-order structures, but the exact role played in this process by protein domains is not well understood. In this study we show the formation of different higher-order structures of the receptor-type phosphatase PTPRJ, detected in HEK293A cells transfected with different PTPRJ expression constructs. In the plasma membrane PTPRJ forms dimers detectable by treatment with the cross-linking reagent BS(3) (bis[sulfosuccinimidyl]suberate). However, other PTPRJ complexes, dependent on the formation of disulfide bonds, are detected by treatment with the oxidant agent H(2)O(2) or by a mutation Asp872Cys, located in the eighth fibronectin type III domain of PTPRJ. A deletion in the eighth fibronectin domain of PTPRJ impairs its dimerization in the plasma membrane and increases the formation of PTPRJ complexes dependent on disulfide bonds that remain trapped in the cytoplasm. The deletion mutant maintains the catalytic activity but is unable to carry out inhibition of proliferation on HeLa cells, achieved by the wild type form, since it does not reach the plasma membrane. Therefore, the intact structure of the eighth fibronectin domain of PTPRJ is critical for its localization in plasma membrane and biological function.

The relationship between resolution of asymptomatic bacterial vaginosis and spontaneous preterm birth in fetal fibronectin-positive women.

PubMed

Hendler, Israel; Andrews, William W; Carey, Christopher J; Klebanoff, Mark A; Noble, William D; Sibai, Baha M; Hillier, Sharon L; Dudley, Donald; Ernest, Joseph M; Leveno, Kenneth J; Wapner, Ronald; Iams, Jay D; Varner, Michael; Moawad, Atef; Miodovnik, Menachem; O'Sullivan, Mary J; Van Dorsten, Peter J

2007-11-01

The purpose of this study was to determine the impact of persistent bacterial vaginosis (BV) on the occurrence of spontaneous preterm birth (SPB) in women who test positive for fetal fibronectin. This is a secondary analysis of a subset of pregnant women who tested positive for BV and fetal fibronectin between 16(0/7) and 25(6/7) weeks of gestation and who participated in randomized placebo controlled trials of antibiotic therapy. Nugent's criteria were used for the diagnosis of BV. Patients were reassessed for the presence of BV after treatment. The rate of SPB at <34 weeks of gestation was analyzed on the basis of treatment mode and BV status at the follow-up visit. The primary studies included a total of 3285 women. A subset of 215 women met the criteria for this analysis. Seventy-seven of 100 patients (77%) in the antibiotics group vs 33 of the 115 patients (28.7%) in the placebo group became BV negative (P < .0001). The rate of SPB at <34 weeks of gestation was lower for BV resolution compared with persistent BV (0 vs 5.7%, respectively; P = .01). In women who tested positive for fetal fibronectin and BV, resolution of BV is associated with less SPB before 34 weeks of gestation.

A simple in vitro model for investigating epithelial/mesenchymal interactions: keratinocyte inhibition of fibroblast proliferation and fibronectin synthesis.

PubMed

Harrison, Caroline A; Dalley, Andrew J; Mac Neil, Sheila

2005-01-01

Hypertrophic scarring and graft contracture are major causes of morbidity after burn injuries. It is well established that application of a split-thickness skin graft reduces scarring and contraction, and cultured epithelial autografts have a similar effect. To investigate the influence of keratinocytes on fibroblast proliferation and fibronectin synthesis, we used an in vitro separated co-culture model in which epithelial sheets were cultured above fibroblast monolayers without physical contact. We also investigated the response of fibroblasts to keratinocyte-conditioned medium (KCM) obtained from confluent and subconfluent keratinocyte monolayers. Both cultured epithelial sheets, composed of adherent fully confluent keratinocytes, and their conditioned medium, reduced fibroblast proliferation. However, KCM from subconfluent keratinocytes stimulated fibroblast proliferation at low concentrations while inhibiting it at higher concentrations, indicating that keratinocytes can produce both mitogenic and growth-inhibiting factors for fibroblasts. KCM, but not epithelial sheet co-culture, also inhibited fibroblast fibronectin synthesis. This indicates regulation of fibroblast phenotype by soluble factors released by the keratinocyte and also suggests that there is a dialogue between keratinocytes and fibroblasts with respect to fibronectin production. We conclude that this separated co-culture model is a simple way to study epithelial/mesenchymal communication particularly with respect to the role of the fibroblast in wound healing.

Upregulation of adhesion complex proteins and fibronectin by human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin).

PubMed

Phan, T T; Allen, J; Hughes, M A; Cherry, G; Wojnarowska, F

2000-01-01

The fresh leaves and extract of the plant Chromolaena odorata are a traditional herbal treatment in developing countries for burns, soft tissue wounds and skin infections. We have previously shown that the extract had an effect on the growth and proliferation of keratinocytes and fibroblasts in culture. This study has demonstrated that Eupolin extract increased expression of several components of the adhesion complex and fibronectin by human keratinocytes. Using indirect immunofluorescence we found increased expression (dose-dependent) of laminin 5, laminin 1, collagen IV, and fibronectin. The expression of the b1 and b4 integrins was upregulated by the extract at low concentrations (0.1 and 1 microg/ml), but the expression was decreased at higher doses of Eupolin (10 microg-150 microg/ml). A number of clinical studies carried out by Vietnamese and international medical investigators have demonstrated the efficacy of this extract on the wound healing process. In this study we have shown that Eupolin stimulated the expression of many proteins of the adhesion complex and fibronectin by human keratinocytes. The adhesion complex proteins are essential to stabilise epithelium and this effect could contribute to the clinical efficacy of Eupolin in healing.

Vitronectin as a Micromanager of Cell Response in Material-Driven Fibronectin Nanonetworks.

PubMed

Cantini, Marco; Gomide, Karina; Moulisova, Vladimira; González-García, Cristina; Salmerón-Sánchez, Manuel

2017-09-01

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation.

The Agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain.

PubMed

Pader, Vera; James, Ellen H; Painter, Kimberley L; Wigneshweraraj, Sivaramesh; Edwards, Andrew M

2014-10-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Agr Quorum-Sensing System Regulates Fibronectin Binding but Not Hemolysis in the Absence of a Functional Electron Transport Chain

PubMed Central

Pader, Vera; James, Ellen H.; Painter, Kimberley L.; Wigneshweraraj, Sivaramesh

2014-01-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. PMID:25092909

T-helper 2 Cytokines, Transforming Growth Factor β1, and Eosinophil Products Induce Fibrogenesis and Alter Muscle Motility in Patients with Eosinophilic Esophagitis

PubMed Central

Rieder, Florian; Nonevski, Ilche; Ma, Jie; Ouyang, Zhufeng; West, Gail; Protheroe, Cheryl; DePetris, Giovanni; Schirbel, Anja; Lapinski, James; Goldblum, John; Bonfield, Tracey; Lopez, Rocio; Harnett, Karen; Lee, James; Hirano, Ikuo; Falk, Gary; Biancani, Piero; Fiocchi, Claudio

2014-01-01

BACKGROUND & AIMS Patients with eosinophilic esophagitis (EoE) often become dysphagic from the combination of organ fibrosis and motor abnormalities. We investigated mechanisms of dysphagia, assessing the response of human esophageal fibroblasts (HEF), muscle cells (HEMC), and esophageal muscle strips to eosinophil-derived products. METHODS Biopsies were collected via endoscopy from the upper, middle and lower thirds of the esophagus of 18 patients with EoE and 21 individuals undergoing endoscopy for other reasons (controls). Primary cultures of esophageal fibroblasts and muscle cells were derived from 12 freshly resected human esophagectomy specimens. Eosinophil distribution was investigated by histologic analyses of full-thickness esophageal tissue. Active secretion of EoE-related mediators was assessed from medium underlying mucosal biopsy cultures. We quantified production of fibronectin and collagen I by HEF and HEMC in response to eosinophil products. We also measured expression of ICAM1 and VCAM1 by, and adhesion of human eosinophils to, HEF and HEMC. Eosinophil products were tested in an esophageal muscle contraction assay. RESULTS Activated eosinophils were present in all esophageal layers. Significantly higher concentrations of eosinophil-related mediators were spontaneously secreted in mucosal biopsies from patients with EoE than controls. Exposure of HEF and HEMC to increasing concentrations of eosinophil products or co-culture with eosinophils caused HEF and HEMC to increase secretion of fibronectin and collagen I; this was inhibited by blocking transforming growth factor (TGF)β1 and p38 mitogen-activated protein kinase (MAKP) signaling. Eosinophil binding to HEF and HEMC increased following incubation of mesenchymal cells with eosinophil-derived products, and decreased following blockade of TGFβ1 and p38MAPK blockade. Eosinophil products reduced electrical field-induced contraction of esophageal muscle strips, but not acetylcholine-induced contraction. CONCLUSION In an analysis of tissues samples from patients with EoE, we linked the presence and activation state of eosinophils in EoE with altered fibrogenesis and motility of esophageal fibroblasts and muscle cells. This process might contribute to the development of dysphagia. PMID:24486052

T-helper 2 cytokines, transforming growth factor β1, and eosinophil products induce fibrogenesis and alter muscle motility in patients with eosinophilic esophagitis.

PubMed

Rieder, Florian; Nonevski, Ilche; Ma, Jie; Ouyang, Zhufeng; West, Gail; Protheroe, Cheryl; DePetris, Giovanni; Schirbel, Anja; Lapinski, James; Goldblum, John; Bonfield, Tracey; Lopez, Rocio; Harnett, Karen; Lee, James; Hirano, Ikuo; Falk, Gary; Biancani, Piero; Fiocchi, Claudio

2014-05-01

Patients with eosinophilic esophagitis (EoE) often become dysphagic from the combination of organ fibrosis and motor abnormalities. We investigated mechanisms of dysphagia, assessing the response of human esophageal fibroblasts (HEFs), human esophageal muscle cells (HEMCs), and esophageal muscle strips to eosinophil-derived products. Biopsy specimens were collected via endoscopy from the upper, middle, and lower thirds of the esophagus of 18 patients with EoE and 21 individuals undergoing endoscopy for other reasons (controls). Primary cultures of esophageal fibroblasts and muscle cells were derived from 12 freshly resected human esophagectomy specimens. Eosinophil distribution was investigated by histologic analyses of full-thickness esophageal tissue. Active secretion of EoE-related mediators was assessed from medium underlying mucosal biopsy cultures. We quantified production of fibronectin and collagen I by HEF and HEMC in response to eosinophil products. We also measured the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by, and adhesion of human eosinophils to, HEFs and HEMCs. Eosinophil products were tested in an esophageal muscle contraction assay. Activated eosinophils were present in all esophageal layers. Significantly higher concentrations of eosinophil-related mediators were secreted spontaneously in mucosal biopsy specimens from patients with EoE than controls. Exposure of HEFs and HEMCs to increasing concentrations of eosinophil products or co-culture with eosinophils caused HEFs and HEMCs to increase secretion of fibronectin and collagen I; this was inhibited by blocking transforming growth factor β1 and p38 mitogen-activated protein kinase signaling. Eosinophil binding to HEFs and HEMCs increased after incubation of mesenchymal cells with eosinophil-derived products, and decreased after blockade of transforming growth factor β1 and p38 mitogen-activated protein kinase blockade. Eosinophil products reduced electrical field-induced contraction of esophageal muscle strips, but not acetylcholine-induced contraction. In an analysis of tissues samples from patients with EoE, we linked the presence and activation state of eosinophils in EoE with altered fibrogenesis and motility of esophageal fibroblasts and muscle cells. This process might contribute to the development of dysphagia. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasan, Nazarul; Hu, Chuan, E-mail: chuan.hu@louisville.edu

2010-01-01

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cellmore » surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.« less

Effect of Antibiotic Exposure on Nugent Score Among Pregnant Women With and Without Bacterial Vaginosis

PubMed Central

Anderson, Brenna; Zhao, Yuan; Andrews, William W.; Dudley, Donald J.; Sibai, Baha; Iams, Jay D.; Wapner, Ronald J.; Varner, Michael W.; Caritis, Steve N.; O’Sullivan, Mary Jo

2013-01-01

Objective To evaluate whether vaginal flora is altered by antibiotic exposure and associated with a risk of preterm birth, particularly among women with initially normal vaginal flora. Methods This was a secondary analysis of a randomized trial of metronidazole and erythromycin for the prevention of preterm birth among women with a positive fetal fibronectin test. Vaginal swabs for Nugent Gram stain score were collected for classification of bacterial vaginosis before and after antibiotic exposure and read at a central laboratory. Change in Nugent score was assessed for women with (score 7 or higher) or without (score lower than 7) bacterial vaginosis. Linear regression analysis evaluated whether change in Nugent score was associated with preterm birth. Results Two-hundred women without and 69 women with bacterial vaginosis had Gram stain performed before and after antibiotic therapy. Median Nugent score for all women declined from 4.0 to 2.0 after antibiotic therapy, p<0.001. Nugent score declined both for those without (from 2.0 to 1.5, p=0.11) and more dramatically, those with bacterial vaginosis (from 8.0 to 3.0, p<0.01). The components of the Nugent score that were affected by antibiotic exposure were similar among women with and without bacterial vaginosis. Antibiotic exposure and the change in Nugent score were unrelated to preterm birth among bacterial vaginosis-negative women. Conclusion Antibiotic exposure is not associated with preterm birth and does not worsen Nugent score among women with normal vaginal flora and positive fetal fibronectin. PMID:21422854

Intestinal decontamination inhibits TLR4 dependent fibronectin mediated crosstalk between stellate cells and endothelial cells in liver fibrosis in mice

PubMed Central

Zhu, Qiang; Zou, Li; Jagavelu, Kumaravelu; Simonetto, Douglas A.; Huebert, Robert C.; Jiang, Zhi-Dong; DuPont, Herbert L.; Shah, Vijay H.

2012-01-01

Background/Aims Liver fibrosis is associated with angiogenesis and leads to portal hypertension. Certain antibiotics reduce complications of liver failure in humans, however, effect of antibiotics on the pathologic alterations of the disease are not fully understood. The aim of this study was to test whether the non-absorbable antibiotic rifaximin could attenuate fibrosis progression and portal hypertension in vivo, and explore potential mechanisms in vitro. Methods Effect of rifaximin on portal pressure, fibrosis, and angiogenesis was examined in wild type and toll like receptor 4 (TLR4) mutant mice after bile duct ligation (BDL). In vitro studies were carried out to evaluate the effect of the bacterial product and TLR agonist, lipopolysaccharide (LPS) on paracrine interactions between hepatic stellate cells (HSC) and liver endothelial cells (LEC) that lead to fibrosis and portal hypertension. Results Portal pressure, fibrosis, and angiogenesis were significantly lower in BDL mice receiving rifaximin compared to BDL mice receiving vehicle. Studies in TLR4 mutant mice confirmed that the effect of rifaximin was dependent on LPS/TLR4 pathway. Fibronectin (FN) was increased in BDL liver and was reduced by rifaximin administration and thus was explored further in vitro as a potential mediator of paracrine interactions of HSC and LEC. In vitro, LPS promoted FN production from HSC. Furthermore, HSC-derived FN promoted LEC migration and angiogenesis. Conclusion These studies expand our understanding of the relationship of intestinal microbiota with fibrosis development by identifying FN as a TLR4 dependent mediator of the matrix and vascular changes that characterize cirrhosis. PMID:22173161

Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis

PubMed Central

Seehusen, Frauke; Al-Azreg, Seham A.; Raddatz, Barbara B.; Haist, Verena; Puff, Christina; Spitzbarth, Ingo; Ulrich, Reiner; Baumgärtner, Wolfgang

2016-01-01

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease. PMID:27441688

Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis.

PubMed

Seehusen, Frauke; Al-Azreg, Seham A; Raddatz, Barbara B; Haist, Verena; Puff, Christina; Spitzbarth, Ingo; Ulrich, Reiner; Baumgärtner, Wolfgang

2016-01-01

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease.

ECM Proteins Glycosylation and Relation to Diabetes

NASA Astrophysics Data System (ADS)

Pernodet, Nadine; Bloomberg, Ayla; Sood, Vandana; Slutsky, Lenny; Ge, Shouren; Clark, Richard; Rafailovich, Miriam

2004-03-01

The chemical modification and crosslinking of proteins by sugar glycosylation contribute to the aging of tissue proteins, and acceleration of this reaction during hyperglycemia is implicated in the pathogenesis of diabetic complications, such as disorder of the wound healing. Advanced glycation endproducts (AGEs) formation and protein crosslinking are irreversible processes that alter the structural and functional properties of proteins, lipid components and nucleic acids. And the mechanism, by which it happens, is not clear. Fibrinogen and fibronectin are plasma proteins, which play a major role in human wound healing. Fibrinogen converts to an insoluble fibrin "gel" following a cut, which eventually forms a clot to prevent blood loss, to direct cell adhesion and migration for forming scars. Fibronectin is a critical protein for cell adhesion and migration in wound healing. The effects of glucose on the binding of these plasma proteins from the extra cellular matrix (ECM) were followed at different concentrations by atomic force microscopy and lateral force modulation to measure the mechanical response of the samples. Glucose solutions (1, 2, and 3mg/mL) were incubated with the protein (100 mg/ml) and silicon (Si) substrates spun with sulfonated polystyrene (SPS) 28% for five days. Data showed that not only the organization of the protein on the surface was affected but also its mechanical properties. At 3 mg/mL glucose, Fn fibers were observed to be harder than those of the control, in good agreement with our hypothesis that glycosylation hardens tissues by crosslinking of proteins in the ECM and might cause fibers to break more easily.

Secretome analysis of human articular chondrocytes unravels catabolic effects of nicotine on the joint.

PubMed

Lourido, Lucía; Calamia, Valentina; Fernández-Puente, Patricia; Mateos, Jesús; Oreiro, Natividad; Blanco, Francisco J; Ruiz-Romero, Cristina

2016-06-01

Osteoarthritis (OA) is a degenerative joint pathology characterized by articular cartilage degradation that lacks from efficient therapy. Since previous epidemiological data show a high controversy regarding the role of smoking in OA, we aimed to evaluate the effects of nicotine (the most physiologically active compound of tobacco) on the joint. Secretome analyses, based on metabolic labeling followed by LC-MALDI-TOF/TOF analysis, were carried out using an in vitro model of articular inflammation (primary human articular chondrocytes treated with interleukin-1β), and also on osteoarthritic cells. ELISA and Western blot assays were performed to verify some of the results. Nineteen proteins were altered by nicotine in the model of articular inflammation, including several cytokines and proteases. We confirmed the increased secretion by nicotine of matrix metalloproteinase 1 and two proposed markers of OA, fibronectin, and chitinase 3-like protein 1. Finally, four components of the extracellular matrix of cartilage were decreased by nicotine in OA chondrocytes. Our data contribute to a better understanding of the molecular mechanisms that are modulated by nicotine in cartilage cells, suggesting a negative effect of this drug on the joint. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fibronectin-based multilayer thin films.

PubMed

Gand, Adeline; Tabuteau, Maud; Chat, Coline; Ladam, Guy; Atmani, Hassan; Van Tassel, Paul R; Pauthe, Emmanuel

2017-08-01

Thin films mimicking the structure and composition of the extra-cellular matrix (ECM) are potentially attractive as biomaterials for cell contacting applications. Layer-by-layer (LbL) assembly of a biological polycation, poly(l-lysine) (PLL), and a common ECM protein, fibronectin (Fn), was employed here to construct nanoscale, ECM mimicking films. Incremental film thickness and interfacial charge magnitude are observed to diminish with layer number, resulting in sub-linear film growth scaling and saturation after about 10 layers. Infrared spectroscopy and electron microscopy together reveal the formation of Fn containing aggregates, whose presence correlates with diminished charge reversal and suppressed LbL assembly. PLL-Fn films induce a significantly greater murine MC3T3-E1 pre-osteoblastic cell proliferation, while maintaining a much higher proportion of Fn in the molecular (as opposed to fibrillar) state, compared to a Fn monolayer, suggesting the enhanced Fn content of these ECM-mimicking films to significantly, and positively, affect cell behavior. Copyright © 2017 Elsevier B.V. All rights reserved.

Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate

NASA Technical Reports Server (NTRS)

Fong, J. H.; Ingber, D. E.

1996-01-01

We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

Small Molecule Inhibitors Target the Tissue Transglutaminase and Fibronectin Interaction

PubMed Central

Yakubov, Bakhtiyor; Chen, Lan; Belkin, Alexey M.; Zhang, Sheng; Chelladurai, Bhadrani; Zhang, Zhong-Yin; Matei, Daniela

2014-01-01

Tissue transglutaminase (TG2) mediates protein crosslinking through generation of ε−(γ-glutamyl) lysine isopeptide bonds and promotes cell adhesion through interaction with fibronectin (FN) and integrins. Cell adhesion to the peritoneal matrix regulated by TG2 facilitates ovarian cancer dissemination. Therefore, disruption of the TG2-FN complex by small molecules may inhibit cell adhesion and metastasis. A novel high throughput screening (HTS) assay based on AlphaLISA™ technology was developed to measure the formation of a complex between His-TG2 and the biotinylated FN fragment that binds TG2 and to discover small molecules that inhibit this protein-protein interaction. Several hits were identified from 10,000 compounds screened. The top candidates selected based on >70% inhibition of the TG2/FN complex formation were confirmed by using ELISA and bioassays measuring cell adhesion, migration, invasion, and proliferation. In conclusion, the AlphaLISA bead format assay measuring the TG2-FN interaction is robust and suitable for HTS of small molecules. One compound identified from the screen (TG53) potently inhibited ovarian cancer cell adhesion to FN, cell migration, and invasion and could be further developed as a potential inhibitor for ovarian cancer dissemination. PMID:24586660

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

PubMed

Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J; Bateman, John F; Wilson, Richard

2013-05-10

Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.

Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels for regenerative medicine applications.

PubMed

Mirazul Islam, M; Cėpla, Vytautas; He, Chaoliang; Edin, Joel; Rakickas, Tomas; Kobuch, Karin; Ruželė, Živilė; Bruce Jackson, W; Rafat, Mehrdad; Lohmann, Chris P; Valiokas, Ramūnas; Griffith, May

2015-01-01

The implant-host interface is a critical element in guiding tissue or organ regeneration. We previously developed hydrogels comprising interpenetrating networks of recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) as substitutes for the corneal extracellular matrix that promote endogenous regeneration of corneal tissue. To render them functional for clinical application, we have now optimized their composition and thereby enhanced their mechanical properties. We have demonstrated that such optimized RHCIII-MPC hydrogels are suitable for precision femtosecond laser cutting to produce complementing implants and host surgical beds for subsequent tissue welding. This avoids the tissue damage and inflammation associated with manual surgical techniques, thereby leading to more efficient healing. Although we previously demonstrated in clinical testing that RHCIII-based implants stimulated cornea regeneration in patients, the rate of epithelial cell coverage of the implants needs improvement, e.g. modification of the implant surface. We now show that our 500μm thick RHCIII-MPC constructs comprising over 85% water are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, unlike the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30μm wide fibronectin stripes enhanced cell attachment and showed the highest mitotic rates, an effect that potentially can be utilized for faster integration of the implant. We have therefore shown that laboratory-produced mimics of naturally occurring collagen and phospholipids can be fabricated into robust hydrogels that can be laser profiled and patterned to enhance their potential function as artificial substitutes of donor human corneas. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Platelet-Derived Growth Factor-BB Stimulates Fibronectin Gene Expression in Fibroblasts Isolated from Rat Thoracic Aorta

DTIC Science & Technology

1994-06-13

MARYLAND 20814-4799 TEACHING HOSPITALS WALTER REED ARMY MEDtCA L CENTER APPROVAL SHEET NAVAL HOSPITAL. BETHESDA MALCOLM GROW AIR FORCE MEDICAL ...CENTER WILFORD HALL "IR FORCE MEDICAL CENTER Title of Dissertation: "Platelet-derived growth factor-BB stimulates fibronectin gene expression in...fascinating world of basic medical science. His dedication and pursuit of excellence in all facets of his work are standards by which I will guide my own

MENA Promotes Tumor-Intrinsic Metastasis through ECM Remodeling and Haptotaxis.

PubMed

Santiago-Medina, Miguel; Yang, Jing

2016-05-01

Oudin and colleagues report a novel and specific function of MENA in mediating directional migration of breast cancer cells toward a fibronectin gradient of increasing concentration. This MENA-mediated haptotactic response depends on the binding of MENA to the α5β1 integrin receptor, adhesion protein signaling, and fibronectin fibrillogenesis. Cancer Discov; 6(5); 474-6. ©2016 AACRSee related article by Oudin et al., p. 516. ©2016 American Association for Cancer Research.

FbpA, a novel multifunctional Listeria monocytogenes virulence factor.

PubMed

Dramsi, S; Bourdichon, F; Cabanes, D; Lecuit, M; Fsihi, H; Cossart, P

2004-07-01

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.

Inflammatory and fibrotic proteins proteomically identified as key protein constituents in urine and stone matrix of patients with kidney calculi.

PubMed

Boonla, Chanchai; Tosukhowong, Piyaratana; Spittau, Björn; Schlosser, Andreas; Pimratana, Chaowat; Krieglstein, Kerstin

2014-02-15

To uncover whether urinary proteins are incorporated into stones, the proteomic profiles of kidney stones and urine collected from the same patients have to be explored. We employed 1D-PAGE and nanoHPLC-ESI-MS/MS to analyze the proteomes of kidney stone matrix (n=16), nephrolithiatic urine (n=14) and healthy urine (n=3). We identified 62, 66 and 22 proteins in stone matrix, nephrolithiatic urine and healthy urine, respectively. Inflammation- and fibrosis-associated proteins were frequently detected in the stone matrix and nephrolithiatic urine. Eighteen proteins were exclusively found in the stone matrix and nephrolithiatic urine, considered as candidate biomarkers for kidney stone formation. S100A8 and fibronectin, representatives of inflammation and fibrosis, respectively, were up-regulated in nephrolithiasis renal tissues. S100A8 was strongly expressed in infiltrated leukocytes. Fibronectin was over-expressed in renal tubular cells. S100A8 and fibronectin were immunologically confirmed to exist in nephrolithiatic urine and stone matrix, but in healthy urine they were undetectable. Conclusion, both kidney stones and urine obtained from the same patients greatly contained inflammatory and fibrotic proteins. S100A8 and fibronectin were up-regulated in stone-baring kidneys and nephrolithiatic urine. Therefore, inflammation and fibrosis are suggested to be involved in the formation of kidney calculi. Copyright © 2013 Elsevier B.V. All rights reserved.

The effect of coimmobilizing heparin and fibronectin on titanium on hemocompatibility and endothelialization.

PubMed

Li, Guicai; Yang, Ping; Qin, Wei; Maitz, Manfred F; Zhou, Shuo; Huang, Nan

2011-07-01

Currently available cardiovascular implants, such as heart valves and stents, exhibit suboptimal biocompatibility because of the incomplete endothelialization and sequential thrombosis formation especially after a long-term implantation. To improve the blood compatibility and endothelialization simultaneously and ensure the long-term effect of the cardiovascular implants, a technique of combining electrostatic interaction and coimmobilization was developed to form heparin and fibronectin (Hep/Fn) films on aminosilanized titanium (Ti) surfaces. The Hep/Fn coimmobilized films were stable after immersion in PBS for five days, probed by wettability studies and by the release kinetics of heparin and fibronectin. Blood compatibility tests showed that the coimmobilized Hep/Fn films displayed lower hemolysis rate, prolonged blood coagulation time, higher AT III binding density, less platelets activation and aggregation, and less fibrinogen conformational change compared with Ti surface. Endothelial cells (ECs) seeding and fibronectin bioactivity results showed more attached and proliferated ECs and exposed cell-binding sites on the Hep/Fn immobilized samples than that on Ti surfaces. Thus, the Hep/Fn coimmobilized films kept excellent bioactivity even after immersion in PBS for five days. Systemic evaluation suggests that the coimmobilization of Hep/Fn complex improves the blood compatibility and promotes the endothelialization simultaneously. We envisage that this method will provide a potential and effective selection for biomaterials surface modification of cardiovascular implants. Copyright © 2011 Elsevier Ltd. All rights reserved.

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy.

PubMed

Sackey-Aboagye, Bridget; Olsen, Abby L; Mukherjee, Sarmistha M; Ventriglia, Alexander; Yokosaki, Yasuyuki; Greenbaum, Linda E; Lee, Gi Yun; Naga, Hani; Wells, Rebecca G

2016-01-01

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9β1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN.

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy

PubMed Central

Sackey-Aboagye, Bridget; Olsen, Abby L.; Mukherjee, Sarmistha M.; Ventriglia, Alexander; Yokosaki, Yasuyuki; Greenbaum, Linda E.; Lee, Gi Yun; Naga, Hani

2016-01-01

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9β1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN. PMID:27741254

Antisense GLUT-1 protects mesangial cells from glucose induction of GLUT-1 and fibronectin expression.

PubMed

Heilig, C W; Kreisberg, J I; Freytag, S; Murakami, T; Ebina, Y; Guo, L; Heilig, K; Loberg, R; Qu, X; Jin, Y; Henry, D; Brosius, F C

2001-04-01

A stable clone of rat mesangial cells expressing antisense GLUT-1 (i.e., MCGT1AS cells) was developed to protect them from high glucose exposure. GLUT-1 protein was reduced 50%, and the 2-deoxy-[(3)H]glucose uptake rate was reduced 33% in MCGT1AS. MCLacZ control cells and MCGT1 GLUT-1-overexpressing cells were used for comparisons. In MCLacZ, 20 mM D-glucose increased GLUT-1 transcription 90% vs. no increase in MCGT1AS. Glucose (8 mM) and 12 mM xylitol [a hexose monophosphate (HMP) shunt substrate] did not stimulate GLUT-1 transcription. An 87% replacement of the standard 8 mM D-glucose with 3-O-methylglucose reduced GLUT-1 transcription 80%. D-Glucose (20 mM) increased fibronectin mRNA and protein by 47 and 100%, respectively, in MCLacZ vs. no increases in MCGT1AS. Fibronectin synthesis was elevated 48% in MCGT1 and reduced 44% in MCGT1AS. We conclude that 1) transcription of GLUT-1 in response to D-glucose depends on glucose metabolism, although not through the HMP shunt, and 2) antisense GLUT-1 treatment of mesangial cells blocks D-glucose-induced GLUT-1 and fibronectin expression, thereby demonstrating a protective effect that could be beneficial in the setting of diabetes.

Ligand-induced Epitope Masking

PubMed Central

Mould, A. Paul; Askari, Janet A.; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A.; Humphries, Martin J.

2016-01-01

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. PMID:27484800

Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells.

PubMed

Innocentin, Silvia; Guimarães, Valeria; Miyoshi, Anderson; Azevedo, Vasco; Langella, Philippe; Chatel, Jean-Marc; Lefèvre, François

2009-07-01

Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA(+)), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA(+) and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA(+)) or its fibronectin binding domains C and D (L. lactis CD(+)). L. lactis FnBPA(+) and L. lactis InlA(+) showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD(+) was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA(+) or L. lactis InlA(+). Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.

Effects of Coating a Titanium Alloy with Fibronectin on the Expression of Osteoblast Gene Markers in the MC3T3 Osteoprogenitor Cell Line

PubMed Central

Rapuano, Bruce E.; Hackshaw, Kyle M.; Schniepp, Hannes C.; MacDonald, Daniel E.

2013-01-01

Purpose A number of environmental and patient-related factors contribute to implant failure. A significant fraction of these failures can be attributed to limited osseointegration resulting from poor bone healing responses. The overall goal of this study was to determine whether surface treatment of a titanium-aluminum-vanadium alloy (Ti-6Al-4V) implant material with a biomimetic protein coating could promote the differentiation of attached osteoblastic cells. The specific aims of the study were to investigate whether osteoprogenitor cells cultured on a rigorously cleaned implant specimen showed a normal pattern of differentiation and whether preadsorbed fibronectin accelerated or enhanced osteoblast differentiation. Materials and Methods Ti-6Al-4V disks were rigorously cleaned, passivated in nitric acid, and dry heat–sterilized; some of the disks were then coated with 1 nmol/L fibronectin. MC3T3 osteoprogenitor cells were then cultured on the pretreated disks for several weeks. Quantitative real-time polymerase chain reaction was performed to measure changes over time in the mRNA levels of osteoblast genes. Results Fibronectin increased the peak expression of all analyzed osteoblast gene markers. “Early” genes that normally mark the proliferative phase (0 to 10 days) of osteoblastic development showed peak expression within the first 10 days after cell attachment to the titanium alloy. In contrast, “late” genes that normally mark the differentiation (10 to 20 days) and mineralization (20 to 36 days) phases of osteoblastogenesis achieved peak expression only after approximately 3 to 4 weeks of culture. Conclusions Osteoprogenitors cultured on a rigorously cleaned Ti-6Al-4V alloy were found to demonstrate a normal pattern of osteoblast differentiation. Preadsorbed fibronectin was observed to stimulate osteoblast differentiation during the mineralization phase of osteoblastogenesis. PMID:23057020

Immunohistochemical studies of wound healing after monopolar electrocautery and ultrasound submucosal inferior nasal turbinate reduction in sheep.

PubMed

Nousia, C S; Gouveris, H; Giatromanolaki, A; Katotomichelakis, M; Ypsilantis, P; Riga, M; Sivridis, E; Watelet, J B; van Cauwenberge, P; Danielides, V

2013-06-01

Extracellular matrix (ECM) proteins such as fibronectin and collagen III, enzymes such as matrix metalloproteinases and macrophages have been demonstrated to intervene in nasal and paranasal sinuses wound healing. To compare concentration of ECM proteins, enzymes and the recruitment of macrophages during wound repair after monopolar electrocautery in contrast with ultrasound submucosal surgical tissue reduction of inferior nasal turbinate (INT) tested in sheep. Prospective controlled study in sheep. Immunostaining for collagen III, fibronectin, CD68 and matrix metalloproteinase-9 (MMP9) was applied in tissue specimens of INT mucosa after monopolar electrocoagulation (MEC) and ultrasound tissue reduction (UTR). Twelve INTs were studied 1, 3 and 8 weeks post-operatively in each interventional group (MEC and UTR) and 5 INTs were studied in animals of the control group (without surgery). The immunoreactivity was quantitatively graded between 0% to 100% immunoreactivity by a blinded senior pathologist. At the end of the study period collagen III, fibronectin and MMP9 were increased in both groups compared to the levels of the control group. When compared to control group, CD68 immunoreactivity was found higher in MEC group but not in UTR group. Fibronectin subepithelial immunoreactivity exhibited a substantial negative correlation with mucosal epithelial cell necrosis, a substantial positive correlation with fibrosis in MEC-treated specimens and a significant positive correlation with sinusoid engorgement in UTR-treated specimens. Collagen III tissue immunoreactivity showed a particularly significant negative correlation with sinusoid engorgement in MEC-treated specimens. Correlation of fibronectin and collagen III immunoreactivity to histopathologic findings suggests different ECM repair processes between MEC and UTR turbinate tissue reduction. The use of CD68 and MMP9 provides additional clues to the mode of actions of these techniques and to the molecular and cellular events of the nasal mucosa wound healing process.

Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus Aureus

PubMed Central

Hirschhausen, Nina; Schlesier, Tim; Peters, Georg; Heilmann, Christine

2012-01-01

Background Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. Methodology/Principal Findings Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. Conclusions/Significance We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections. PMID:22768285

Characterization of the modular design of the autolysin/adhesin Aaa from Staphylococcus aureus.

PubMed

Hirschhausen, Nina; Schlesier, Tim; Peters, Georg; Heilmann, Christine

2012-01-01

Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections.

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation

PubMed Central

Casillas-Ituarte, Nadia N.; Cruz, Carlos H. B.; Lins, Roberto D.; DiBartola, Alex C.; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F. T.; Sierra-Hernández, M. Roxana; Lower, Steven K.

2017-01-01

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2–0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. PMID:28400484

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation.

PubMed

Casillas-Ituarte, Nadia N; Cruz, Carlos H B; Lins, Roberto D; DiBartola, Alex C; Howard, Jessica; Liang, Xiaowen; Höök, Magnus; Viana, Isabelle F T; Sierra-Hernández, M Roxana; Lower, Steven K

2017-05-26

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands ( e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/ k off ) and dissociation constants ( K d = k off / k on ), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I ( K d app = 0.2-0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide ( K d app = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Altered Gravity and Early Heart Development in Culture

NASA Technical Reports Server (NTRS)

Wiens, Darrell J.; Lwigale, P.; Denning, J.

1996-01-01

The macromolecules comprising the cytoskeleton and extracellular matrix of cells may be sensitive to gravitation. Since early development of organs depends on dynamic interactions across cell surfaces, altered gravity may disturb development. We investigated this possibility for heart development. Previous studies showed that the extracellular matrix glycoprotein fibronectin (Fn) is necessary for normal heart development. We cultured precardiac tissue explants in a high aspect ratio bioreactor vessel (HARV) to simulate microgravity. We observed tissue morphology, contraction, and Fn distribution by immunolocalization in HARV rotated and control (lxg) explants, cultured 18 hr. We also measured Fn amount by immunoassay. Explants in HARV were rotated at 6 rpm to achieve continuous freefall. Thirty-five of 37 control, but only 1 of 37 matched rotated explants exhibited contractions. Tissue architecture was identical. Immunolocalization of Fn showed remarkable differences which may be related to the development of contractions. The Fn staining in the HARV explants was less intense in all areas. Areas of linear staining along epithelia were present but shorter, and there was less intercellular staining in both mesenchymal tissue and myocardium. Initial immunoassay results of 5 matched pairs of explants showed a 22% reduction in total tissue Fn in the HARV rotated samples. Our results indicate that altered gravity in the HARV reduced the amount and distribution of Fn, as assessed by two independent criteria. This was correlated with a reduction in the development of contractile activity.

Fibronectin Matrix Remodeling in the Regulation of the Inflammatory Response within the Lung: An Early Step in Lung Cancer Progression

DTIC Science & Technology

2011-09-01

such as that which occurs in chronic obstructive pulmonary disease (COPD) and emphysema , is associated with increased risk of lung cancer. These...effect of the fibronectin III-1c peptide on the expression of inflammatory genes by human lung fibroblasts, lung cancer cells, and pulmonary ...CXCR2 in bleomycin-induced pulmonary inflammation and fibrosis. Am J Respir Cell Mol Biol. 2009; 40:410-21. 7. Barnes PJ. New therapies for chronic

Cytochemical Changes in Hepatocytes of Rats with Endotoxemia and Sepsis: Localization of Fibronectin, Calcium, and Enzymes

DTIC Science & Technology

1988-01-01

rate was deposited predominantly on the outer surfaces of in the pathogn~nesis of endotoxernia and septic shock. The the RER of hepatocytes. in additron...es; and (c) tin it- the basal (perisinusoidal) surfaces and in the cisternae G-6-Pase activity. LPS treatment also leads to reduced num- of tough...by Kupffer cells (Cook et al., 1985’ Gut et al., sue fibronectin results in widening intercellular junctions anci in- 198: ~nkaa ndIwsk,18

Exosome secretion promotes chemotaxis of cancer cells.

PubMed

Sung, Bong Hwan; Weaver, Alissa M

2017-03-04

Migration of cells toward chemical cues, or chemotaxis, is important for many biologic processes such as immune defense, wound healing and cancer metastasis. Although chemotaxis is thought to occur in cancer cells, it is less well characterized than chemotaxis of professional immune cells such as neutrophils. Here, we show that cancer cell chemotaxis relies on secretion of exosome-type extracellular vesicles. Migration of fibrosarcoma cells toward a gradient of exosome-depleted serum was diminished by knockdown of the exosome secretion regulator Rab27a. Rescue experiments in which chemotaxis chambers were coated with purified extracellular vesicles demonstrate that exosomes but not microvesicles affect both speed and directionality of migrating cells. Chamber coating with purified fibronectin and fibronectin-depleted exosomes demonstrates that the exosome cargo fibronectin promotes cell speed but cannot account for the role of exosomes in promoting directionality of fibrosarcoma cell movement during chemotaxis. These experiments indicate that exosomes contain multiple motility-promoting cargoes that contribute to different aspects of cell motility.

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes.

PubMed

Uemura, Akiyoshi; Kusuhara, Sentaro; Wiegand, Stanley J; Yu, Ruth T; Nishikawa, Shin-ichi

2006-02-01

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes.

Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes

PubMed Central

Uemura, Akiyoshi; Kusuhara, Sentaro; Wiegand, Stanley J.; Yu, Ruth T.; Nishikawa, Shin-Ichi

2006-01-01

In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes. PMID:16424942

Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

PubMed Central

Liberio, Michelle S.; Sadowski, Martin C.; Soekmadji, Carolina; Davis, Rohan A.; Nelson, Colleen C.

2014-01-01

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement. PMID:25375165

Origin of tumor-promoter released fibronectin in fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burrous, B.A.; Wolf, G.

1986-05-01

Previous work from the laboratory showed that the chemical tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated release of the cell surface glycoprotein, fibronectin (FN) from human lung fibroblasts (HLF), leading to depletion of cell surface FN, while FN synthesis is not altered by TPA. To further investigate the mechanism(s) by which TPA stimulates FN release, two types of experiments were performed. In the first, HLF were pulsed with /sup 35/S-methionine-labeled medium with or without TPA. In the second, cell-surface proteins were labeled by iodination (/sup 125/I) and then incubated in unlabeled medium with or without TPA. In both cases, the fate ofmore » labeled FN was followed over 12 hr. The /sup 35/S-meth-labeled HLF showed a rapid loss of labeled FN, first into a small, highly-labeled pool of cell surface FN (1 hr), later into the medium (4 hr or longer). Specific activities showed that this small pool in the cell surface turned over rapidly. TPA treatment resulted in more rapid movement of /sup 35/S-meth pulse-labeled FN to the cell surface and into the medium than in control cells. TPA thus affected the fate of intracellular FN. TPA treatment of HLF also resulted in more rapid removal of /sup 125/I-cell surface-labeled FN into the medium than in control cells. Thus, TPA affects the fate of preexisting cell surface FN in HLF. From these results, they hypothesize that TPA has two separate effects: it stimulates depletion of preexisting intracellular FN during the first hr of treatment, and it stimulates release of preexisting cell surface FN over all treatment times.« less

pERK1/2 Peripheral Recruitment and Filopodia Protrusion Augment Oligodendrocyte Progenitor Cell Migration: Combined Effects of PDGF-A and Fibronectin.

PubMed

Tripathi, Ashutosh; Parikh, Zalak S; Vora, Parvez; Frost, Emma E; Pillai, Prakash P

2017-03-01

Oligodendrocyte progenitor cell (OPC) migration is critical for effective myelination of the central nervous system. Not only during normal myelination but also during remyelination, the growth factors (GFs) and extracellular matrix (ECM) protein affect the OPC migration. Studies showed the altered levels of GFs and ECM in the demyelinating lesions. In our earlier studies, we have shown that the effect of platelet-derived growth factor alpha (PDGF-A) on OPC migration is dose- and time-dependent. In that we have shown that the physiological concentration (1 ng/ml) of PDGF-A was unable to induce OPC migration at transient exposure (30 min). However, the involvement of ECM in the regulation of PDGF-A mediated OPC migration was not clear. In the present study, we have used fibronectin (FN) as ECM. PDGF-A and FN have similar and overlapping intracellular signaling pathways including the extracellular regulated kinases 1 and 2 (ERK1/2). Here we demonstrate how physiological concentration of PDGF-A combines with FN to augment OPC migration in vitro. The present study is first of its kind to show the importance of the synergistic effects of PDGF-A and FN on peripheral recruitment of phosphorylated/activated ERK1/2 (pERK1/2), actin-pERK1/2 co-localization, and filopodia formation, which are essential for the enhanced OPC migration. These findings were further confirmed by ERK1/2 inhibition studies, using the pharmacological inhibitor U0126. An understanding of these complex interactions may lead to additional strategies for transplanting genetically modified OPCs to repair widespread demyelinated lesions.

A Novel Antifibrotic Mechanism of Nintedanib and Pirfenidone. Inhibition of Collagen Fibril Assembly.

PubMed

Knüppel, Larissa; Ishikawa, Yoshihiro; Aichler, Michaela; Heinzelmann, Katharina; Hatz, Rudolf; Behr, Jürgen; Walch, Axel; Bächinger, Hans Peter; Eickelberg, Oliver; Staab-Weijnitz, Claudia A

2017-07-01

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix, in particular, collagens. Two IPF therapeutics, nintedanib and pirfenidone, decelerate lung function decline, but their underlying mechanisms of action are poorly understood. In this study, we sought to analyze their effects on collagen synthesis and maturation at important regulatory levels. Primary human fibroblasts from patients with IPF and healthy donors were treated with nintedanib (0.01-1.0 μM) or pirfenidone (100-1,000 μM) in the absence or presence of transforming growth factor-β1. Effects on collagen, fibronectin, FKBP10, and HSP47 expression, and collagen I and III secretion, were analyzed by quantitative polymerase chain reaction and Western blot. The appearance of collagen fibrils was monitored by scanning electron microscopy, and the kinetics of collagen fibril assembly was assessed using a light-scattering approach. In IPF fibroblasts, nintedanib reduced the expression of collagen I and V, fibronectin, and FKBP10 and attenuated the secretion of collagen I and III. Pirfenidone also down-regulated collagen V but otherwise showed fewer and less pronounced effects. By and large, the effects were similar in donor fibroblasts. For both drugs, electron microscopy of IPF fibroblast cultures revealed fewer and thinner collagen fibrils compared with untreated controls. Finally, both drugs dose-dependently delayed fibril formation of purified collagen I. In summary, both drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in down-regulating profibrotic gene expression and collagen secretion. Importantly, both drugs inhibited collagen I fibril formation and caused a reduction in and an altered appearance of collagen fibril bundles, representing a completely novel mechanism of action for both drugs.

Pirfenidone may revert the epithelial-to-mesenchymal transition in human lung adenocarcinoma.

PubMed

Kurimoto, Ryota; Ebata, Takahiro; Iwasawa, Shunichiro; Ishiwata, Tsukasa; Tada, Yuji; Tatsumi, Koichiro; Takiguchi, Yuichi

2017-07-01

The epithelial-to-mesenchymal transition (EMT) in cancer is associated with invasion, metastasis and chemoresistance. Recent studies have revealed the increased expression of programmed death-ligand 1 (PD-L1) in cells undergoing EMT. The underlying mechanism of EMT involves transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2). Pirfenidone and the known EMT-suppressor nintedanib suppress pulmonary fibrosis partially through suppression of TGF-β. The present study aimed to determine whether pirfenidone has the potential to induce EMT-reversion, using nintedanib as a reference. The human lung adenocarcinoma cell lines A-549, HCC-827, and PC-9 were treated with TGF-β and FGF-2 to induce EMT. The EMT-induced cells were further treated with pirfenidone or nintedanib. Phenotypic alterations associated with EMT were assessed by examining the following: i) The expression levels of E-cadherin, vimentin, fibronectin and slug, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescent immunohistochemistry; ii) cell motility via wound-healing assays; and iii) the expression of PD-L1 using RT-qPCR. The combination of TGF-β and FGF-2 successfully induced EMT in all three cell lines, characterized by a significant reduction in E-cadherin expression in the A-549 and HCC-827 cells, increased expression levels of vimentin, fibronectin, slug and PD-L1, and increased cell motility in all three cell lines. Pirfenidone and nintedanib reverted all of these phenotypes, with the exception of unaltered E-cadherin expression in all three cell lines, and inconsistent expression of vimentin in the HCC-827 and PC-9 cells. Thus, pirfenidone and nintedanib have the ability to induce EMT-reversion in human lung adenocarcinoma.

Beneficial effects of previous exercise training on renal changes in streptozotocin-induced diabetic female rats

PubMed Central

Amaral, Liliany S de Brito; Silva, Fernanda A; Correia, Vicente B; Andrade, Clara EF; Dutra, Bárbara A; Oliveira, Márcio V; de Magalhães, Amélia CM; Volpini, Rildo A; Seguro, Antonio C; Coimbra, Terezila M

2016-01-01

This study evaluated the effects of aerobic exercise performed both previously and after the induction of diabetes mellitus on changes of renal function and structure in streptozotocin-induced diabetic rats. Female wistar rats were divided into five groups: sedentary control (C + Se); trained control (C + Ex); sedentary diabetic (D + Se); trained diabetic (D + Ex) and previously trained diabetic (D + PEx). The previous exercise consisted of treadmill running for four weeks before the induction of diabetes mellitus. After induction of diabetes mellitus with streptozotocin, the D + PEx, D + Ex and C + Ex groups were submitted to eight weeks of aerobic exercise. At the end of the training protocol, we evaluate the serum glucose, insulin and 17β-estradiol levels, renal function and structure, proteinuria, and fibronectin, collagen IV and transforming growth factor beta 1 (TGF-β1) renal expressions. Induction of diabetes mellitus reduced the insulin and did not alter 17β-estradiol levels, and exercise did not affect any of these parameters. Previous exercise training attenuated the loss of body weight, the blood glucose, the increase of glomerular filtration rate and prevented the proteinuria in the D + PEx group compared to D + Se group. Previous exercise also reduced glomerular hypertrophy, tubular and glomerular injury, as well as the expressions of fibronectin and collagen IV. These expressions were associated with reduced expression of TGF-β1. In conclusion, our study shows that regular aerobic exercise especially performed previously to induction of diabetes mellitus improved metabolic control and has renoprotective action on the diabetic kidney. PMID:26490345

Beneficial effects of previous exercise training on renal changes in streptozotocin-induced diabetic female rats.

PubMed

Amaral, Liliany S de Brito; Silva, Fernanda A; Correia, Vicente B; Andrade, Clara E F; Dutra, Bárbara A; Oliveira, Márcio V; de Magalhães, Amélia C M; Volpini, Rildo A; Seguro, Antonio C; Coimbra, Terezila M; Soares, Telma de J

2016-02-01

This study evaluated the effects of aerobic exercise performed both previously and after the induction of diabetes mellitus on changes of renal function and structure in streptozotocin-induced diabetic rats. Female wistar rats were divided into five groups: sedentary control (C + Se); trained control (C + Ex); sedentary diabetic (D + Se); trained diabetic (D + Ex) and previously trained diabetic (D + PEx). The previous exercise consisted of treadmill running for four weeks before the induction of diabetes mellitus. After induction of diabetes mellitus with streptozotocin, the D + PEx, D + Ex and C + Ex groups were submitted to eight weeks of aerobic exercise. At the end of the training protocol, we evaluate the serum glucose, insulin and 17β-estradiol levels, renal function and structure, proteinuria, and fibronectin, collagen IV and transforming growth factor beta 1 (TGF-β1) renal expressions. Induction of diabetes mellitus reduced the insulin and did not alter 17β-estradiol levels, and exercise did not affect any of these parameters. Previous exercise training attenuated the loss of body weight, the blood glucose, the increase of glomerular filtration rate and prevented the proteinuria in the D + PEx group compared to D + Se group. Previous exercise also reduced glomerular hypertrophy, tubular and glomerular injury, as well as the expressions of fibronectin and collagen IV. These expressions were associated with reduced expression of TGF-β1. In conclusion, our study shows that regular aerobic exercise especially performed previously to induction of diabetes mellitus improved metabolic control and has renoprotective action on the diabetic kidney. © 2016 by the Society for Experimental Biology and Medicine.

Fibronectin and bovine serum albumin adsorption and conformational dynamics on inherently conducting polymers: a QCM-D study.

PubMed

Molino, Paul J; Higgins, Michael J; Innis, Peter C; Kapsa, Robert M I; Wallace, Gordon G

2012-06-05

Quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to characterize the adsorption of the model proteins, bovine serum albumin (BSA) and fibronectin (FN), to polypyrrole doped with dextran sulfate (PPy-DS) as a function of DS loading and surface roughness. BSA adsorption was greater on surfaces of increased roughness and was above what could be explained by the increase in surface area alone. Furthermore, the additional mass adsorbed on the rough films was concomitant with an increase in the rigidity of the protein layer. Analysis of the dynamic viscoelastic properties of the protein adlayer reveal BSA adsorption on the rough films occurs in two phases: (1) arrival and initial adsorption of protein to the polymer surface and (2) postadsorption molecular rearrangement to a more dehydrated and compact conformation that facilitates further recruitment of protein to the polymer interface, likely forming a multilayer. In contrast, FN adsorption was independent of surface roughness. However, films prepared from solutions containing the highest concentration of DS (20 mg/mL) demonstrated both an increase in adsorbed mass and adlayer viscoelasticity. This is attributed to the higher DS loading in the conducting polymer film resulting in presentation of a more hydrated molecular structure indicative of a more unfolded and bioactive conformation. Modulating the redox state of the PPy-DS polymers was shown to modify both the adsorbed mass and viscoelastic nature of FN adlayers. An oxidizing potential increased both the total adsorbed mass and the adlayer viscoelasticity. Our findings demonstrate that modification of polymer physicochemical and redox condition alters the nature of protein-polymer interaction, a process that may be exploited to tailor the bioactivity of protein through which interactions with cells and tissues may be controlled.

R-Ras Regulates Migration through an Interaction with Filamin A in Melanoma Cells

PubMed Central

Gawecka, Joanna E.; Griffiths, Genevieve S.; Ek-Rylander, Barbro; Ramos, Joe W.; Matter, Michelle L.

2010-01-01

Background Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins. Methods and Findings We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin β1, β2 and β7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaΔ3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly. Conclusions These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration. PMID:20585650

Occurrence of fibronectin-fibrin complexes in plasma of patients with multimorbidity due to the inflamm-aging phenomenon.

PubMed

Pupek, Małgorzata; Pawłowicz, Robert; Lindner, Karolina; Krzyżanowska-Gołąb, Dorota; Lemańska-Perek, Anna; Panaszek, Bernard; Kątnik-Prastowska, Iwona

2016-05-01

Multimorbidity is the co-occurrence of chronic diseases associated with low-grade chronic inflammation of connective tissue. Frequency of occurrence and relative amounts of fibronectin (FN) complexes with fibrin (FN-fibrin) and FN monomer were analyzed in 130 plasma samples of 18 to 94-year-old multimorbid patients in relation to concentrations of FN and extra domain A (EDA)-FN, and C-reactive protein (CRP) as well as to age, number of coexisting chronic diseases and presence of specified diseases. Immunoblotting revealed, besides FN dimer, the presence of FN monomer, and 750-, 1000-, and 1300-kDa FN-fibrin complexes in the multimorbid plasmas. The FN-fibrin complexes appeared more frequently and in higher relative amounts, but FN monomer less frequently and in a lower relative amount in the groups of elderly multimorbid patients, with a higher number of coexisting diseases and with dominance of cardiovascular diseases and osteoarthrosis, and with CRP concentration of 3-5mg/l. In contrast, the normal plasma contained only the FN-fibrin complex of 750 kDa in a lower relative amount, but with an increasing amount with normal aging. Moreover, FN concentration increased and EDA-FN decreased with the number of co-existing diseases and aging of patients, although both concentration values were lower than in the age-matched normal groups. FN concentration was the lowest in the exacerbation of a chronic disease and EDA-FN in the stable chronic disease groups. The alterations in plasma FN molecular status were associated with micro-inflammation and micro-coagulation, as well as multimorbidity of subjects and their physiological aging. Copyright © 2016 Elsevier Inc. All rights reserved.

Dermal matrix proteins initiate re-epithelialization but are not sufficient for coordinated epidermal outgrowth in a new fish skin culture model.

PubMed

Matsumoto, Reiko; Sugimoto, Masazumi

2007-02-01

We have established a new culture system to study re-epithelialization during fish epidermal wound healing. In this culture system, fetal bovine serum (FBS) stimulates the epidermal outgrowth of multi-cellular layers from scale skin mounted on a coverslip, even when cell proliferation is blocked. The rate of outgrowth is about 0.4 mm/h, and at 3 h after incubation, the area occupied by the epidermal sheet is nine times larger than the area of the original scale skin. Cells at the bottom of the outgrowth show a migratory phenotype with lamellipodia, and "purse string"-like actin bundles have been found over the leading-edge cells with polarized lamellipodia. In the superficial cells, re-development of adherens junctions and microridges has been detected, together with the appearance and translocation of phosphorylated p38 MAPK into nuclear areas. Thus, this culture system provides an excellent model to study the mechanisms of epidermal outgrowth accompanied by migration and re-differentiation. We have also examined the role of extracellular matrix proteins in the outgrowth. Type I collagen or fibronectin stimulates moderate outgrowth in the absence of FBS, but development of microridges and the distribution of phosphorylated p38 MAPK are attenuated in the superficial cells. In addition, the leading-edge cells do not have apparent "purse string"-like actin bundles. The outgrowth stimulated by FBS is inhibited by laminin. These results suggest that dermal substrates such as type I collagen and fibronectin are able to initiate epidermal outgrowth but require other factors to enhance such outgrowth, together with coordinated alterations in cellular phenotype.

CCL18 synergises with high concentrations of glucose in stimulating fibronectin production in human renal tubuloepithelial cells.

PubMed

Montero, Rosa M; Bhangal, Gurjeet; Pusey, Charles D; Frankel, Andrew H; Tam, Frederick W K

2016-09-29

Diabetic nephropathy is the leading cause of end stage kidney disease worldwide. The pathogenesis of this disease remains elusive and multiple factors have been implicated. These include the effects of hyperglycaemia, haemodynamic and metabolic factors, and an inflammatory process that stimulates cellular signalling pathways leading to disease progression and severe fibrosis. Fibronectin (Fn) is an important protein of the extracellular matrix that is essential in fibrosis and its presence in increased amounts has been identified in the kidney in diabetic nephropathy. Proximal tubuloepithelial (HK-2) cells were stimulated with high glucose (30 mM D-glucose) or glycated albumin (500 μg/mmol) + 4 mM D-glucose or their controls, Mannitol (26 mM + 4 mM D-glucose) and 4 mM D-glucose, respectively. Following 48 h of stimulation the supernatant was collected and MTT [3-(4,5-dimethylthiazole-2,5-diphenyltetrazolium bromide] assay performed to assess cell viability. HK-2 cells were also stimulated in the above environments with recombinant CCL18 (rCCL18) or MCP-1 (rMCP-1) for 48 h with quantification of Fn levels using ELISA. Co-stimulation of HK-2 cells with high concentrations of glucose and rCCL18 significantly increased Fn (p < 0.001), in comparison to high concentrations of glucose alone. HK-2 cells stimulated with glycated albumin consistently produced Fn and this did not alter following co-stimulation with rCCL18 or rMCP-1. This study demonstrates how stimulation with a specific chemokine CCL18 in high glucose upregulates the production of Fn from proximal tubuloepithelial cells. This may be relevant to the development of renal fibrosis in diabetic nephropathy.

Fibronectin matrix assembly suppresses dispersal of glioblastoma cells.

PubMed

Sabari, Joshua; Lax, Daniel; Connors, Daniel; Brotman, Ian; Mindrebo, Eric; Butler, Christine; Entersz, Ildiko; Jia, Dongxuan; Foty, Ramsey A

2011-01-01

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12-15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5β1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal.

[Changes in the thrombophilic status in patients with pre-eclampsia].

PubMed

Baptista-González, H A; Rosenfeld-Mann, F; Saavedra-Trejo, M R; Castro-López, J L; Peñuela-Olaya, M A

1999-04-01

The object of this study was to evaluate the changes in fibrinolysis and clotting inhibitors in patients with preeclampsia and to describe the connection between preeclampsia and blood pressure values. Two groups of pregnant women were prospectively studied at delivery: group 1 women without preeclampsia and group 2 patients with preeclampsia. The variables that were registered are: diastolic blood pressure (DBP), systolic blood pressure (SBP), mean blood pressure (MBP), hemoglobin (Hb), platelet count (Plt), lupus like inhibitor, anticardiolipin antibodies (ACA), antinuclear antibodies (ANA), fibronectina, D dimer, protein S (PS), protein C (PC) and vo Willebrand factor (vWF). 62 pregnant women were included. The patients of group 2 presented high values of Hb (p 0.01), fibronectin (p 0.0001), D-dimer (p 0.01) and lower PC (p 0.04). We found an association between fibronectin and higher values of SBP, DBP, MBP and Hb (p 0.0007) versus lower values of VFW and PC (p 0.002). The low values of total PS were associated with high D-dimer and SBP results (p 0.04 and 0.002 respectively). All patients were ACA/ANA negative. In preclampsia there is a increased hemoconcentration and drop in clotting inhibitors (PC), without fibrinolytic compensatory response (lower D-dimer) and remarked vasopressive effect (hig fibronectin). This changes depend on the stratification of blood pressure. Th SBP and MBP values depend on the haemodynamic changes (Hb, fibronectin), while the increase in DBP expresses a non compensated thrombophilic state.

Amino acid sequence of the human fibronectin receptor

PubMed Central

1987-01-01

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+- binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors. PMID:2958481

Influenza viral neuraminidase primes bacterial coinfection through TGF-β-mediated expression of host cell receptors.

PubMed

Li, Ning; Ren, Aihui; Wang, Xiaoshuang; Fan, Xin; Zhao, Yong; Gao, George F; Cleary, Patrick; Wang, Beinan

2015-01-06

Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-β. Because TGF-β can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-β during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-β signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-β. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-β signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-β, leading to increased bacterial loading in the lungs. Our results suggest that TGF-β and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.

Dual-functional biomimetic materials: nonfouling poly(carboxybetaine) with active functional groups for protein immobilization.

PubMed

Zhang, Zheng; Chen, Shengfu; Jiang, Shaoyi

2006-12-01

We introduce a dual-functional biocompatible material based on zwitterionic poly(carboxybetaine methacrylate) (polyCBMA), which not only highly resists protein adsorption/cell adhesion, but also has abundant functional groups convenient for the immobilization of biological ligands, such as proteins. The dual-functional properties are unique to carboxybetaine moieties and are not found in other nonfouling moieties such as ethylene glycol, phosphobetaine, and sulfobetaine. The unique properties are demonstrated in this work by grafting a polyCBMA polymer onto a surface or by preparing a polyCBMA-based hydrogel. PolyCBMA brushes with a thickness of 10-15 nm were grafted on a gold surface using the surface-initiated atom transfer radical polymerization method. Protein adsorption was analyzed using a surface plasmon resonance sensor. The surface grafted with polyCBMA very largely prevented the nonspecific adsorption of three test proteins, that is, fibrinogen, lysozyme, and human chorionic gonadotropin (hCG). The immobilization of anti-hCG on the surface resulted in the specific binding of hCG while maintaining a high resistance to nonspecific protein adsorption. Transparent polyCBMA-based hydrogel disks were decorated with immobilized fibronectin. Aortic endothelial cells did not bind to the polyCBMA controls, but appeared to adhere well and spread on the fibronectin-modified surface. With their dual functionality and biomimetic nature, polyCBMA-based materials are very promising for their applications in medical diagnostics, biomaterials/tissue engineering, and drug delivery.

Crystal structure of the second fibronectin type III (FN3) domain from human collagen α1 type XX.

PubMed

Zhao, Jingfeng; Ren, Jixia; Wang, Nan; Cheng, Zhong; Yang, Runmei; Lin, Gen; Guo, Yi; Cai, Dayong; Xie, Yong; Zhao, Xiaohong

2017-12-01

Collagen α1 type XX, which contains fibronectin type III (FN3) repeats involving six FN3 domains (referred to as the FN#1-FN#6 domains), is an unusual member of the fibril-associated collagens with interrupted triple helices (FACIT) subfamily of collagens. The results of standard protein BLAST suggest that the FN3 repeats might contribute to collagen α1 type XX acting as a cytokine receptor. To date, solution NMR structures of the FN#3, FN#4 and FN#6 domains have been determined. To obtain further structural evidence to understand the relationship between the structure and function of the FN3 repeats from collagen α1 type XX, the crystal structure of the FN#2 domain from human collagen α1 type XX (residues Pro386-Pro466; referred to as FN2-HCXX) was solved at 2.5 Å resolution. The crystal structure of FN2-HCXX shows an immunoglobulin-like fold containing a β-sandwich structure, which is formed by a three-stranded β-sheet (β1, β2 and β5) packed onto a four-stranded β-sheet (β3, β4, β6 and β7). Two consensus domains, tencon and fibcon, are structural analogues of FN2-HCXX. Fn8, an FN3 domain from human oncofoetal fibronectin, is the closest structural analogue of FN2-HCXX derived from a naturally occurring sequence. Based solely on the structural similarity of FN2-HCXX to other FN3 domains, the detailed functions of FN2-HCXX and the FN3 repeats in collagen α1 type XX cannot be identified.

Functional Na+ Channels in Cell Adhesion probed by Transistor Recording

PubMed Central

Schmidtner, Markus; Fromherz, Peter

2006-01-01

Cell membranes in a tissue are in close contact to each other, embedded in the extracellular matrix. Standard electrophysiological methods are not able to characterize ion channels under these conditions. Here we consider the area of cell adhesion on a solid substrate as a model system. We used HEK 293 cells cultured on fibronectin and studied the activation of NaV1.4 sodium channels in the adherent membrane with field-effect transistors in a silicon substrate. Under voltage clamp, we compared the transistor response with the whole-cell current. We observed that the extracellular voltage in the cell-chip contact was proportional to the total membrane current. The relation was calibrated by alternating-current stimulation. We found that Na+ channels are present in the area of cell adhesion on fibronectin with a functionality and a density that is indistinguishable from the free membrane. The experiment provides a basis for studying selective accumulation and depletion of ion channels in cell adhesion and also for a development of cell-based biosensoric devices and neuroelectronic systems. PMID:16227504

Selective reversible inhibition of autophagy in hypoxic breast cancer cells promotes pulmonary metastasis

PubMed Central

Dower, Christopher M.; Bhat, Neema; Wang, Edward W.; Wang, Hong-Gang

2016-01-01

Autophagy influences how cancer cells respond to nutrient deprivation and hypoxic stress, two hallmarks of the tumor microenvironment (TME). In this study, we explored the impact of autophagy on the pathophysiology of breast cancer cells, using a novel hypoxia-dependent, reversible dominant negative strategy to regulate autophagy at the cellular level within the TME. Suppression of autophagy via hypoxia-induced expression of the kinase-dead unc-51 like autophagy activating kinase (ULK1) mutant K46N increased lung metastases in MDA-MB-231 xenograft mouse models. Consistent with this effect, expressing a dominant-negative mutant of ULK1 or ATG4b or a ULK1-targeting shRNA facilitated cell migration in vitro. Functional proteomic and transcriptome analysis revealed that loss of hypoxia-regulated autophagy promotes metastasis via induction of the fibronectin integrin signaling axis. Indeed, loss of ULK1 function increased fibronectin deposition in the hypoxic TME. Together, our results indicated that hypoxia-regulated autophagy suppresses metastasis in breast cancer by preventing tumor fibrosis. These results also suggest cautions in the development of autophagy-based strategies for cancer treatment. PMID:28115361

Utilizing Fibronectin Integrin-Binding Specificity to Control Cellular Responses

PubMed Central

Bachman, Haylee; Nicosia, John; Dysart, Marilyn; Barker, Thomas H.

2015-01-01

Significance: Cells communicate with the extracellular matrix (ECM) protein fibronectin (Fn) through integrin receptors on the cell surface. Controlling integrin–Fn interactions offers a promising approach to directing cell behavior, such as adhesion, migration, and differentiation, as well as coordinated tissue behaviors such as morphogenesis and wound healing. Recent Advances: Several different groups have developed recombinant fragments of Fn that can control epithelial to mesenchymal transition, sequester growth factors, and promote bone and wound healing. It is thought that these physiological responses are, in part, due to specific integrin engagement. Furthermore, it has been postulated that the integrin-binding domain of Fn is a mechanically sensitive switch that drives binding of one integrin heterodimer over another. Critical Issues: Although computational simulations have predicted the mechano-switch hypothesis and recent evidence supports the existence of varying strain states of Fn in vivo, experimental evidence of the Fn integrin switch is still lacking. Future Directions: Evidence of the integrin mechano-switch will enable the development of new Fn-based peptides in tissue engineering and wound healing, as well as deepen our understanding of ECM pathologies, such as fibrosis. PMID:26244106

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

A clinicopathological study of the expression of extracellular matrix components in urothelial carcinoma.

PubMed

Ioachim, Elli; Michael, Michalis; Stavropoulos, Nicolaos E; Kitsiou, Evangelia; Salmas, Marios; Malamou-Mitsi, Vasiliki

2005-03-01

To measure the immunohistochemical expression of the extracellular matrix (ECM) components tenascin, fibronectin, collagen type IV and laminin in urothelial carcinomas, and to correlate their expression with clinicopathological features to clarify the prognostic value of these molecules and their role in tumour progression. Tumour specimens obtained during transurethral resection of bladder tumour (TURBT) from 103 patients (82 men and 2 1 women, mean age 66.7 years, range 27-89) were studied retrospectively. The expression of tenascin, fibronectin, collagen type IV and laminin was correlated with clinicopathological features (tumour grade and stage, multiplicity, simultaneous in situ component, the proliferative activity as estimated by the two proliferation associated indices, Ki-67 and proliferating cell nuclear antigen, the recurrence rate, and the progression of invading tumour). Specimens investigated for tenascin expression from patients with superficial bladder cancers were categorized into 28 treated by TURBT only and 53 who had TURBT followed by intravesical instillations of interferon. Cytoplasmic tenascin expression was detected in tumour cells in 20% of specimens. Tenascin was expressed in the tumour stroma in 76% of specimens, and was positively correlated with tumour grade and stage. Stromal tenascin expression was positively correlated with proliferative activity, and with the expression of fibronectin and collagen type IV. Fibronectin was expressed in the tumour stroma in 89% of specimens and was positively correlated with tumour stage, proliferative activity, and expression of collagen type IV and laminin. Collagen type IV was expressed in 93% of specimens, and was positively correlated with tumour grade and stage. Laminin was expressed in 78% of specimens and had no significant correlation with the clinicopathological features. Patients treated with TURBT alone and who had low levels of tenascin had a longer tumour-free interval than those with high levels of tenascin. Levels of tenascin might be valuable for predicting the risk of early recurrence. The expression of tenascin, fibronectin and collagen type IV seems to be correlated with more aggressive tumour behaviour. Furthermore, their interrelationships could indicate that they are involved in the remodelling of bladder cancer tissue, probably influencing tumour progression.

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

PubMed

Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

2013-11-01

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular adhesion. These data indicate that Wnt/Glypican4/Frizzled signaling regulates ECM assembly through effects on cadherin-mediated cell cohesion. Together, our results demonstrate that zebrafish Vangl2/Prickle1a and non-canonical Wnt/Frizzled signaling have opposing effects on ECM organization underlying PCP and gastrulation cell movements. © 2013 Elsevier Inc. All rights reserved.

The Effect of Gravity Fields on Cellular Gene Expression

NASA Technical Reports Server (NTRS)

Hughes-Fulford, Millie

1999-01-01

Early theoretical analysis predicted that microgravity effects on the isolated cell would be minuscule at the subcellular level; however, these speculations have not proven true in the real world. Astronauts experience a significant bone and muscle loss in as little as 2 weeks of spaceflight and changes are seen at the cellular level soon after exposure to microgravity. Changes in biological systems may be primarily due to the lack of gravity and the resulting loss of mechanical stress on tissues and cells. Recent ground and flight studies examining the effects of gravity or mechanical stress on cells demonstrate marked changes in gene expression when relatively small changes in mechanical forces or gravity fields were made. Several immediate early genes (IEG) like c-fos and c-myc are induced by mechanical stimulation within minutes. In contrast, several investigators report that the absence of mechanical forces during space flight result in decreased sera response element (SRE) activity and attenuation of expression of IEGs such as c-fos, c-jun and cox-2 mRNAs. Clearly, these early changes in gene expression may have long term consequences on mechanically sensitive cells. In our early studies on STS-56, we reported four major changes in the osteoblast; 1) prostaglandin synthesis in flight, 2) changes in cellular morphology, 3) altered actin cytoskeleton and 4) reduced osteoblast growth after four days exposure to microgravity. Initially, it was believed that changes in fibronectin (FN) RNA, FN protein synthesis or subsequent FN matrix formation might account for the changes in cytoskeleton and/ or reduction of growth. However our recent studies on Biorack (STS-76, STS-81 and STS-84), using ground and in-flight 1-G controls, demonstrated that fibronectin synthesis and matrix formation were normal in microgravity. In addition, in our most recent Biorack paper, our laboratory has documented that relative protein synthesis and mRNA synthesis are not changed after 24 hours exposure to microgravity. We did, however, find significant changes in osteoblast gene expression of IEGs, c-fos and cox-2 in microgravity exposure as compared to ground and in-flight 1-G controls. Subsequent ground studies suggest that the molecular mechanism underlying these changes may involve prostaglandin c-AMP receptors (EPs) and/or subsequent alteration of intracellular signaling in the absence of gravity.

Cartilage proteoglycans inhibit fibronectin-mediated adhesion

NASA Astrophysics Data System (ADS)

Rich, A. M.; Pearlstein, E.; Weissmann, G.; Hoffstein, S. T.

1981-09-01

Normal tissues and organs show, on histological examination, a pattern of cellular and acellular zones that is characteristic and unique for each organ or tissue. This pattern is maintained in health but is sometimes destroyed by disease. For example, in mobile joints, the articular surfaces consist of relatively acellular hyaline cartilage, and the joint space is enclosed by a capsule of loose connective tissue with a lining of fibroblasts and macrophages. In the normal joint these cells are confined to the synovial lining and the articular surface remains acellular. In in vitro culture, macrophages and their precursor monocytes are very adhesive, and fibroblasts can migrate and overgrow surfaces such as collagen or plastic used for tissue culture. The fibroblasts adhere to collagen by means of fibronectin, which they synthesize and secrete1. Because the collagen of cartilage is capable of binding serum fibronectin2 and fibronectin is present in cartilage during its development3, these cells should, in theory, slowly migrate from the synovial lining to the articular surface. It is their absence from the articular cartilage in normal circumstances, and then presence in such pathological states as rheumatoid arthritis, that is striking. We therefore set out to determine whether a component of cartilage could prevent fibroblast adherence in a defined adhesion assay. As normal cartilage is composed of 50% proteoglycans and 50% collagen by dry weight4, we tested the possibility that the proteoglycans in cartilage inhibit fibroblast adhesion to collagen. We present here evidence that fibroblast spreading and adhesion to collagenous substrates is inhibited by cartilage proteoglycans.

Importance of adhesins in the recurrence of pharyngeal infections caused by Streptococcus pyogenes.

PubMed

Wozniak, Aniela; Scioscia, Natalia; Geoffroy, Enrique; Ponce, Iván; García, Patricia

2017-04-01

Pharyngo-amygdalitis is the most common infection caused by Streptococcus pyogenes (S. pyogenes). Reinfection with strains of different M types commonly occurs. However, a second infection with a strain of the same M type can still occur and is referred to as recurrence. We aimed to assess whether recurrence of S. pyogenes could be associated to erythromycin resistance, biofilm formation or surface adhesins like fibronectin-binding proteins and pilus proteins, both located in the fibronectin-binding, collagen-binding, T-antigen (FCT) region. We analyed clinical isolates of S. pyogenes obtained from children with multiple positive cultures of throat swabs. We analysed potential associations between M types, clonal patterns, biofilm production and FCT types with their capacity of producing a recurrent infection. We genetically defined recurrence as an infection with the same M type (same strain) and reinfection as an infection with a different M type. No differences were observed between recurrent and reinfection isolates in relation to erythromycin resistance, presence and number of domains of prtF1 gene, and biofilm formation capacity; the only significant difference was the higher frequency of FCT-4 type among recurrent isolates. However, when all the factors that could contribute to recurrence (erythromycin resistance, biofilm production, presence of prtF1 gene and FCT-4 type) were analysed together, we observed that recurrent isolates have a higher number of factors than reinfection isolates. Recurrence seems not to be associated with biofilm formation. However, pili and fibronectin-binding proteins could be associated with recurrence because FCT-4 isolates which harbour two fibronectin-binding proteins are more frequent among recurrent isolates.

Ablation of lens epithelial cells with a laser photolysis system: Histopathology, ultrastructure, and immunochemistry

PubMed Central

Mamalis, Nick; Grossniklaus, Hans E.; Waring, George O.; Werner, Liliana; Brubaker, Jacob; Davis, Don; Espandar, Ladan; Walker, Rudolf; Thyzel, Reinhardt

2010-01-01

PURPOSE To evaluate efficacy of a neodymium:YAG (Nd:YAG) laser photolysis system in removing lens epithelial cells (LECs) and characterize the effect of the laser on laminin and fibronectin involved in LEC adhesion and migration. METHODS Cadaver eyes were evaluated using the Miyake technique. The lenses were removed with phacoemulsification. The modified Nd:YAG laser was used to clean the LECs from the capsule. Only the fornix was cleaned in some eyes and the anterior subcapsular area in other eyes. Some areas were not treated and acted as controls. Standard irrigation/aspiration (I/A) removal of LECs was performed in additional eyes. The eyes were analyzed using light microscopy and immunohistochemical staining. RESULTS Histopathologic evaluation showed that the laser removed the LECs from the anterior lens capsule and from the fornix. Immunohistochemical staining showed fibronectin and laminin staining in the untreated areas that was absent in the treated areas. Standard I/A removal of the LECs showed absence of cells but persistent laminin and fibronectin. Electron microscopy showed epithelial cells in untreated areas with an absence of the LECs and debris in treated areas. CONCLUSIONS The laser photolysis system removed LECs from the anterior lens capsule and capsule fornix. Along with the cells, laminin, fibronectin, and cell debris remained in the untreated areas but were removed by the treatment. This treatment may be useful in preventing posterior capsule opacification. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned. Additional disclosures are found in the footnotes. PMID:20494774

Prenatal Exposure to Nicotine and Childhood Asthma: Role of Nicotine Acetylcholine Receptors, Neuropeptides and Fibronectin Expression in Lung

DTIC Science & Technology

2008-12-01

of apoptosis , how it might be affected by nicotine, and how it might be involved in lung development. This idea was based on observations generated...Furthermore, we recently found that agents capable of inhibiting apoptosis (zinc and autocarboxylic acid) inhibit lung branching morphogenesis...cell apoptosis in lung development. We discovered that apoptosis is most prominent in pseudoglandular-stage lungs coinciding with the time period of

Mifepristone inhibits extracellular matrix formation in uterine leiomyoma.

PubMed

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

2016-04-01

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

Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

NASA Astrophysics Data System (ADS)

Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

2017-04-01

Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

Current treatment of vocal fold scarring.

PubMed

Hirano, Shigeru

2005-06-01

Vocal fold scarring still remains a therapeutic challenge, with the most problematic issue being the histologic changes that are primarily responsible for altering the viscoelasticity of the vocal fold mucosa. Optimal treatment for vocal fold scarring has not yet been established. To restore or regenerate damaged vocal folds, it is important to investigate the changes to the layer structure of the lamina propria. Tissue engineering and regenerative medicine may provide new strategies for the prevention and treatment of vocal fold scarring. Recent developments in this field are reviewed in the present article. Histologic studies have revealed that hyaluronic acid, fibronectin, decorin, and various other extracellular matrix components, as well as collagen, may contribute to determining the vibratory properties of the vocal fold mucosa. Changes of these molecules are thought to affect the viscoelasticity of the scarred vocal folds. Based on such histologic findings, innovative approaches have been developed, including administration of hyaluronic acid into injured or scarred vocal folds. Other strategies that have recently shown advances include growth factor therapy and cell therapy using stem cells or mature fibroblasts. The effects of these new treatments have not fully been confirmed clinically, but there seems to be great therapeutic potential in such regenerative medical strategies. Recent research has revealed the detailed histologic and rheologic changes related to vocal fold scarring. Based on these findings, various new therapeutic strategies have been developed in animal models using tissue engineering and regenerative medicine. However, no clinical trials have been performed, and more studies are necessary to establish the optimum modality.

Anterior Segment Alterations and Comparative Aqueous Humor Proteomics in the Buphthalmic Rabbit (An American Ophthalmological Society Thesis)

PubMed Central

Edward, Deepak P.; Bouhenni, Rachida

2011-01-01

Purpose To use an integrated proteohistologic approach to gain insight into the anterior segment alterations in the buphthalmic rabbit. Methods Eyes from 2- and 5-year-old buphthalmic and normal rabbits (n=20) were studied histologically. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) of aqueous humor (AH) was used to determine differential protein expression between animal groups. Western blot and immunohistochemistry were performed on selected differentially expressed proteins identified by LC-MS/MS. Results The buphthalmic rabbits manifested a mild clinical phenotype with typical angle anomalies that appeared progressive by histology. Significantly thickened Descemet’s membrane (DM) and anterior lens capsule in all buphthalmic rabbits showed increased fibronectin and collagen-IV immunolabeling. LC-MS/MS applying stringent filtering criteria revealed significant differential expression of several AH proteins in these rabbits. The protein of interest in the 2-year-old group was histidine-rich glycoprotein, and those in the 5-year-old group included alpha-2-HS-glycoprotein, clusterin, apolipoprotein E, interphotoreceptor retinoid-binding protein, transthyretin, cochlin, gelsolin, haptoglobin, hemopexin, and beta-2 microglobulin. The proteomic data for selected proteins was validated by Western blot and immunohistochemistry. A wide range of functional groups were affected by the altered AH proteins. These included extracellular matrix modulation, regulation of apoptosis, oxidative stress, and protein transport. Conclusions Multiple anterior segment alterations were histologically identified in the buphthalmic rabbits that showed progressive changes with age. The differentially expressed AH proteins in these rabbits suggest a multifunctional role for AH in modulating pathologic changes in DM, anterior lens capsule, and the angular meshwork in these animals. PMID:22253484

Stabilization, Rolling, and Addition of Other Extracellular Matrix Proteins to Collagen Hydrogels Improve Regeneration in Chitosan Guides for Long Peripheral Nerve Gaps in Rats.

PubMed

Gonzalez-Perez, Francisco; Cobianchi, Stefano; Heimann, Claudia; Phillips, James B; Udina, Esther; Navarro, Xavier

2017-03-01

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. 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. 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. 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. 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. Copyright © 2017 by the Congress of Neurological Surgeons

Biofunctionalized aligned microgels provide 3D cell guidance to mimic complex tissue matrices.

PubMed

Rose, Jonas C; Gehlen, David B; Haraszti, Tamás; Köhler, Jens; Licht, Christopher J; De Laporte, Laura

2018-05-01

Natural healing is based on highly orchestrated processes, in which the extracellular matrix plays a key role. To resemble the native cell environment, we introduce an artificial extracellular matrix (aECM) with the capability to template hierarchical and anisotropic structures in situ, allowing a minimally-invasive application via injection. Synthetic, magnetically responsive, rod-shaped microgels are locally aligned and fixed by a biocompatible surrounding hydrogel, creating a hybrid anisotropic hydrogel (Anisogel), of which the physical, mechanical, and chemical properties can be tailored. The microgels are rendered cell-adhesive with GRGDS and incorporated either inside a cell-adhesive fibrin or bioinert poly(ethylene glycol) hydrogel to strongly interact with fibroblasts. GRGDS-modified microgels inside a fibrin-based Anisogel enhance fibroblast alignment and lead to a reduction in fibronectin production, indicating successful replacement of structural proteins. In addition, YAP-translocation to the nucleus increases with the concentration of microgels, indicating cellular sensing of the overall anisotropic mechanical properties of the Anisogel. For bioinert surrounding PEG hydrogels, GRGDS-microgels are required to support cell proliferation and fibronectin production. In contrast to fibroblasts, primary nerve growth is not significantly affected by the biomodification of the microgels. In conclusion, this approach opens new opportunities towards advanced and complex aECMs for tissue regeneration. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Novel Potential Interacting Partners of Fibronectin in Spontaneous Animal Model of Interstitial Cystitis

PubMed Central

Treutlein, Gudrun; Dorsch, Roswitha; Euler, Kerstin N.; Hauck, Stefanie M.; Amann, Barbara; Hartmann, Katrin; Deeg, Cornelia A.

2012-01-01

Feline idiopathic cystitis (FIC) is the only spontaneous animal model for human interstitial cystitis (IC), as both possess a distinctive chronical and relapsing character. Underlying pathomechanisms of both diseases are not clearly established yet. We recently detected increased urine fibronectin levels in FIC cases. The purpose of this study was to gain further insight into the pathogenesis by assessing interacting partners of fibronectin in urine of FIC affected cats. Several candidate proteins were identified via immunoprecipitation and mass spectrometry. Considerable changes in FIC conditions compared to physiological expression of co-purified proteins were detected by Western blot and immunohistochemistry. Compared to controls, complement C4a and thioredoxin were present in higher levels in urine of FIC patients whereas loss of signal intensity was detected in FIC affected tissue. Galectin-7 was exclusively detected in urine of FIC cats, pointing to an important role of this molecule in FIC pathogenesis. Moderate physiological signal intensity of galectin-7 in transitional epithelium shifted to distinct expression in transitional epithelium under pathophysiological conditions. I-FABP expression was reduced in urine and urinary bladder tissue of FIC cats. Additionally, transduction molecules of thioredoxin, NF-κB p65 and p38 MAPK, were examined. In FIC affected tissue, colocalization of thioredoxin and NF-κB p65 could be demonstrated compared to absent coexpression of thioredoxin and p38 MAPK. These considerable changes in expression level and pattern point to an important role for co-purified proteins of fibronectin and thioredoxin-regulated signal transduction pathways in FIC pathogenesis. These results could provide a promising starting point for novel therapeutic approaches in the future. PMID:23236492

Telmisartan Therapy Does Not Improve Lymph Node or Adipose Tissue Fibrosis More Than Continued Antiretroviral Therapy Alone.

PubMed

Utay, Netanya S; Kitch, Douglas W; Yeh, Eunice; Fichtenbaum, Carl J; Lederman, Michael M; Estes, Jacob D; Deleage, Claire; Magyar, Clara; Nelson, Scott D; Klingman, Karen L; Bastow, Barbara; Luque, Amneris E; McComsey, Grace A; Douek, Daniel C; Currier, Judith S; Lake, Jordan E

2018-05-05

Fibrosis in lymph nodes may limit CD4+ T-cell recovery, and lymph node and adipose tissue fibrosis may contribute to inflammation and comorbidities despite antiretroviral therapy (ART). We hypothesized that the angiotensin receptor blocker and peroxisome proliferator-activated receptor γ agonist telmisartan would decrease lymph node or adipose tissue fibrosis in treated human immunodeficiency virus type 1 (HIV) infection. In this 48-week, randomized, controlled trial, adults continued HIV-suppressive ART and received telmisartan or no drug. Collagen I, fibronectin, and phosphorylated SMAD3 (pSMAD3) deposition in lymph nodes, as well as collagen I, collagen VI, and fibronectin deposition in adipose tissue, were quantified by immunohistochemical analysis at weeks 0 and 48. Two-sided rank sum and signed rank tests compared changes over 48 weeks. Forty-four participants enrolled; 35 had paired adipose tissue specimens, and 29 had paired lymph node specimens. The median change overall in the percentage of the area throughout which collagen I was deposited was -2.6 percentage points (P = 0.08) in lymph node specimens and -1.3 percentage points (P = .001) in adipose tissue specimens, with no between-arm differences. In lymph node specimens, pSMAD3 deposition changed by -0.5 percentage points overall (P = .04), with no between-arm differences. Telmisartan attenuated increases in fibronectin deposition (P = .06). In adipose tissue, changes in collagen VI deposition (-1.0 percentage point; P = .001) and fibronectin deposition (-2.4 percentage points; P < .001) were observed, with no between-arm differences. In adults with treated HIV infection, lymph node and adipose tissue fibrosis decreased with continued ART alone, with no additional fibrosis reduction with telmisartan therapy.

Adhesive interactions of human multiple myeloma cell lines with different extracellular matrix molecules.

PubMed

Kibler, C; Schermutzki, F; Waller, H D; Timpl, R; Müller, C A; Klein, G

1998-06-01

Multiple myeloma represents a human B cell malignancy which is characterized by a predominant localization of the malignant cell clone within the bone marrow. With the exception of the terminal stage of the disease the myeloma tumor cells do not circulate in the peripheral blood. The bone marrow microenvironment is believed to play an important role in homing, proliferation and terminal differentiation of myeloma cells. Here we have studied the expression of several extracellular matrix (ECM) molecules in the bone marrow of multiple myeloma patients and analyzed their adhesive capacities with four different human myeloma-derived cell lines. All ECM molecules analyzed (tenascin, laminin, fibronectin, collagen types I, III, V and VI) could be detected in bone marrow cryostat sections of multiple myeloma patients. Adhesion assays showed that only laminin, the microfibrillar collagen type VI and fibronectin were strong adhesive components for the myeloma cell lines U266, IM-9, OPM-2 and NCI-H929. Tenascin and collagen type I were only weak adhesive substrates for these myeloma cells. Adhesion to laminin and fibronectin was beta 1-integrin-mediated since addition of anti-beta 1-integrin antibodies could inhibit the binding of the four different cell types to both matrix molecules. In contrast, integrins do not seem to be involved in binding of the myeloma cells to collagen type VI. Instead, inhibition of binding by heparin suggested that membrane-bound heparan sulfate proteoglycans are responsible ligands for binding to collagen type VI. Adhesion assays with several B-cell lines resembling earlier differentiation stages revealed only weak interactions with tenascin and no interactions with collagen type VI, laminin or fibronectin. In summary, the interactions of human myeloma cells with the extracellular matrix may explain the specific retention of the plasma cells within the bone marrow.

Role of extracellular matrix in regulation of staurosporine-induced apoptosis in breast cancer cells.

PubMed

Vasaturo, F; Malacrino, C; Sallusti, E; Coppotelli, G; Birarelli, P; Giuffrida, A; Albonici, L; Simonelli, L; Modesti, A; Modesti, M; Scarpa, S

2005-04-01

Autocrine and paracrine mechanisms modulate the synthesis and secretion of extracellular matrix (ECM); moreover, each component of the ECM is capable of modulating the synthesis and release of other ECM molecules. Therefore, the synthesis of ECM glycoprotein fibronectin and laminin was studied in the human breast cancer cell lines MCF7 and MDA MB 23, plated on different ECM. Our results showed that the cells plated on a fibronectin substrate increased laminin synthesis: this event correlated with an increase in alpha2 and alpha3 integrin subunits. Staurosporine-induced apoptosis was then analyzed in the cell lines plated on different ECM. Staurosporine treatment determined the apoptosis of 35 and 33% respectively of MDA MB 231 and MCF7; these values increased to 60 and 64% in cells plated on laminin, to 48 and 63% in cells plated on fibronectin and to 64 and 69% in cells plated on matrigel. Moreover, staurosporine treatment decreased bcl-2 expression in the cells plated on fibronectin and laminin. Yet, staurosporine treatment determined PARP cleavage and PARP partial disappearance when the cells were plated on matrigel. Finally, a partial loss of function mutant Ras protein that activated only Raf pathway, was expressed in MCF7, in order to identify whether the increase of apoptosis induced by extracellular matrix involved the Raf/MAP kinase pathway. The increase of apoptosis of the cells plated on matrigel suggested that the activation of the Raf pathway is probably involved in the decrease of survival on matrigel. These data demonstrate that the modification of ECM modulates the apoptotic process of breast cancer cells and suggest that it is worthwhile to dissect the role of ECM in the control of apoptotic process.

Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Talamás-Lara, Daniel, E-mail: daniel_talamas@hotmail.com; Talamás-Rohana, Patricia, E-mail: ptr@cinvestav.mx; Fragoso-Soriano, Rogelio Jaime, E-mail: rogelio@fis.cinvestav.mx

Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves amore » dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica. - Highlights: • Striking differences in adhesion to FN between E. histolytica and E. dispar. • A greater degree of cell stiffness in E. histolytica with respect to E. dispar. • E. histolytica but not E. dispar forms regions of close contact with FN. • The actin cytoskeleton is involved in the pathogenicity of E. histolytica.« less

Keratocyte Apoptosis and Not Myofibroblast Differentiation Mark the Graft/Host Interface at Early Time-Points Post-DSAEK in a Cat Model

PubMed Central

Weis, Adam J.; Huxlin, Krystel R.; Callan, Christine L.; DeMagistris, Margaret A.; Hindman, Holly B.

2013-01-01

Purpose To evaluate myofibroblast differentiation as an etiology of haze at the graft-host interface in a cat model of Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK). Methods DSAEK was performed on 10 eyes of 5 adult domestic short-hair cats. In vivo corneal imaging with slit lamp, confocal, and optical coherence tomography (OCT) were performed twice weekly. Cats were sacrificed and corneas harvested 4 hours, and 2, 4, 6, and 9 days post-DSAEK. Corneal sections were stained with the TUNEL method and immunohistochemistry was performed for α-smooth muscle actin (α-SMA) and fibronectin with DAPI counterstain. Results At all in vivo imaging time-points, corneal OCT revealed an increase in backscatter of light and confocal imaging revealed an acellular zone at the graft-host interface. At all post-mortem time-points, immunohistochemistry revealed a complete absence of α-SMA staining at the graft-host interface. At 4 hours, extracellular fibronectin staining was identified along the graft-host interface and both fibronectin and TUNEL assay were positive within adjacent cells extending into the host stroma. By day 2, fibronectin and TUNEL staining diminished and a distinct acellular zone was present in the region of previously TUNEL-positive cells. Conclusions OCT imaging consistently showed increased reflectivity at the graft-host interface in cat corneas in the days post-DSAEK. This was not associated with myofibroblast differentiation at the graft-host interface, but rather with apoptosis and the development of a subsequent acellular zone. The roles of extracellular matrix changes and keratocyte cell death and repopulation should be investigated further as potential contributors to the interface optical changes. PMID:24098706

Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor β.

PubMed

Rawal, S Y; Dabbous, M Kh; Tipton, D A

2012-06-01

Marijuana (Cannabis sativa) use may be associated with gingival enlargement, resembling that caused by phenytoin. Cannabidiol (CBD), a nonpsychotropic Cannabis derivative, is structurally similar to phenytoin. While there are many reports on effects of phenytoin on human gingival fibroblasts, there is no information on effects of Cannabis components on these cells. The objective of this study was to determine effects of CBD on human gingival fibroblast fibrogenic and matrix-degrading activities. Fibroblasts were incubated with CBD in serum-free medium for 1-6 d. The effect of CBD on cell viability was determined by measuring activity of a mitochondrial enzyme. The fibrogenic molecule transforming growth factor β and the extracellular matrix molecule fibronectin were measured by ELISA. Pro-MMP-1 and total MMP-2 were measured by ELISA. Activity of MMP-2 was determined via a colorimetric assay in which a detection enzyme is activated by active MMP-2. Data were analysed using ANOVA and Scheffe's F procedure for post hoc comparisons. Cannabidiol had little or no significant effect on cell viability. Low CBD concentrations increased transforming growth factor β production by as much as 40% (p < 0.001), while higher concentrations decreased it by as much as 40% (p < 0.0001). Cannabidiol increased fibronectin production by as much as approximately 100% (p < 0.001). Lower CBD concentrations increased MMP production, but the highest concentrations decreased production of both MMPs (p < 0.05) and decreased MMP-2 activity (p < 0.02). The data suggest that the CBD may promote fibrotic gingival enlargement by increasing gingival fibroblast production of transforming growth factor β and fibronectin, while decreasing MMP production and activity. © 2011 John Wiley & Sons A/S.

Type of MRI contrast, tissue gadolinium, and fibrosis.

PubMed

Do, Catherine; Barnes, Jeffrey L; Tan, Chunyan; Wagner, Brent

2014-10-01

It has been presupposed that the thermodynamic stability constant (K(therm)) of gadolinium-based MRI chelates relate to the risk of precipitating nephrogenic systemic fibrosis. The present study compared low-K(therm) gadodiamide with high-K(therm) gadoteridol in cultured fibroblasts and rats with uninephrectomies. Gadolinium content was assessed using scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy in paraffin-embedded tissues. In vitro, fibroblasts demonstrated dose-dependent fibronectin generation, transforming growth factor-β production, and expression of activated myofibroblast stress fiber protein α-smooth muscle actin. There were negligible differences with respect to toxicity or proliferation between the two contrast agents. In the rodent model, gadodiamide treatment led to greater skin fibrosis and dermal cellularity than gadoteridol. In the kidney, both contrast agents led to proximal tubule vacuolization and increased fibronectin accumulation. Despite large detectable gadolinium signals in the spleen, skin, muscle, and liver from the gadodiamide-treated group, contrast-induced fibrosis appeared to be limited to the skin and kidney. These findings support the hypothesis that low-K(therm) chelates have a greater propensity to elicit nephrogenic systemic fibrosis and demonstrate that certain tissues are resistant to these effects.

The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes

PubMed Central

Benito-Jardón, Maria; Klapproth, Sarah; Gimeno-LLuch, Irene; Petzold, Tobias; Bharadwaj, Mitasha; Müller, Daniel J; Zuchtriegel, Gabriele; Reichel, Christoph A; Costell, Mercedes

2017-01-01

Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level. DOI: http://dx.doi.org/10.7554/eLife.22264.001 PMID:28092265

EDB Fibronectin Specific Peptide for Prostate Cancer Targeting.

PubMed

Han, Zheng; Zhou, Zhuxian; Shi, Xiaoyue; Wang, Junpeng; Wu, Xiaohui; Sun, Da; Chen, Yinghua; Zhu, Hui; Magi-Galluzzi, Cristina; Lu, Zheng-Rong

2015-05-20

Extradomain-B fibronectin (EDB-FN), one of the oncofetal fibronectin (onfFN) isoforms, is a high-molecular-weight glycoprotein that mediates cell adhesion and migration. The expression of EDB-FN is associated with a number of cancer-related biological processes such as tumorigenesis, angiogenesis, and epithelial-to-mesenchymal transition (EMT). Here, we report the development of a small peptide specific to EDB-FN for targeting prostate cancer. A cyclic nonapeptide, CTVRTSADC (ZD2), was identified using peptide phage display. A ZD2-Cy5 conjugate was synthesized to accomplish molecular imaging of prostate cancer in vitro and in vivo. ZD2-Cy5 demonstrated effective binding to up-regulated EDB-FN secreted by TGF-β-induced PC3 cancer cells following EMT. Following intravenous injections, the targeted fluorescent probe specifically bound to and delineated PC3-GFP prostate tumors in nude mice bearing the tumor xenografts. ZD2-Cy5 also showed stronger binding to human prostate tumor specimens with a higher Gleason score (GS9) compared to those with a lower score (GS 7), with no binding in benign prostatic hyperplasia (BPH). Thus, the ZD2 peptide is a promising strategy for molecular imaging and targeted therapy of prostate cancer.

Micrometer scale spacings between fibronectin nanodots regulate cell morphology and focal adhesions

NASA Astrophysics Data System (ADS)

Horzum, Utku; Ozdil, Berrin; Pesen-Okvur, Devrim

2014-04-01

Cell adhesion to extracellular matrix is an important process for both health and disease states. Surface protein patterns that are topographically flat, and do not introduce other chemical, topographical or rigidity related functionality and, more importantly, that mimic the organization of the in vivo extracellular matrix are desired. Previous work showed that vinculin and cytoskeletal organization are modulated by size and shape of surface nanopatterns. However, quantitative analysis on cell morphology and focal adhesions as a function of micrometer scale spacings of FN nanopatterns was absent. Here, electron beam lithography was used to pattern fibronectin nanodots with micrometer scale spacings on a K-casein background on indium tin oxide coated glass which, unlike silicon, is transparent and thus suitable for many light microscopy techniques. Exposure times were significantly reduced using the line exposure mode with micrometer scale step sizes. Micrometer scale spacings of 2, 4 and 8 μm between fibronectin nanodots proved to modulate cell adhesion through modification of cell area, focal adhesion number, size and circularity. Overall, cell behavior was shown to shift at the apparent threshold of 4 μm spacing. The findings presented here offer exciting new opportunities for cell biology research.

Assembly and remodeling of the fibrillar fibronectin extracellular matrix during gastrulation and neurulation in Xenopus laevis.

PubMed

Davidson, Lance A; Keller, Raymond; DeSimone, Douglas W

2004-12-01

Fibronectin, a major component of the extracellular matrix is critical for processes of cell traction and cell motility. Whole-mount confocal imaging of the three-dimensional architecture of the extracellular matrix is used to describe dynamic assembly and remodeling of fibronectin fibrils during gastrulation and neurulation in the early frog embryo. As previously reported, fibrils first appear under the prospective ectoderm. We describe here the first evidence for regulated assembly of fibrils along the somitic mesoderm/endoderm boundary as well as at the notochord/somitic mesoderm boundary and clearing of fibrils from the dorsal and ventral surfaces of the notochord that occurs over the course of a few hours. As gastrulation proceeds, fibrils are restored to the dorsal surface of the notochord, where the notochord contacts the prospective floor plate. As the neural folds form, fibrils are again remodeled as deep neural plate cells move medially. The process of neural tube closure leaves a region of the ectoderm overlying the neural crest transiently bare of fibrils. Fibrils are assembled surrounding the dorsal surface of the neural tube as the neural tube lumen is restored. Copyright (c) 2004 Wiley-Liss, Inc.

Synergistic effects of BMP-2, BMP-6 or BMP-7 with human plasma fibronectin onto hydroxyapatite coatings: A comparative study.

PubMed

Brigaud, Isabelle; Agniel, Rémy; Leroy-Dudal, Johanne; Kellouche, Sabrina; Ponche, Arnaud; Bouceba, Tahar; Mihailescu, Natalia; Sopronyi, Mihai; Viguier, Eric; Ristoscu, Carmen; Sima, Felix; Mihailescu, Ion N; Carreira, Ana Claudia O; Sogayar, Mari Cleide; Gallet, Olivier; Anselme, Karine

2017-06-01

Design of new osteoinductive biomaterials to reproduce an optimized physiological environment capable of recruiting stem cells and instructing their fate towards the osteoblastic lineage has become a priority in orthopaedic surgery. This work aims at evaluating the bioactivity of BMP combined with human plasma fibronectin (FN/BMP) delivered in solution or coated onto titanium-hydroxyapatite (TiHA) surfaces. Herein, we focus on the comparison of in vitro osteogenic efficacy in mouse C2C12 pre-osteoblasts of three BMP members, namely: BMP-2, BMP-6 and BMP-7. In parallel, we evaluated the molecular binding strength between each BMP with FN using the Surface Plasmon Resonance (SPR) technology. The affinity of BMPs for FN was found totally different and dependent on BMP type. Indeed, the combination of FN with BMP-2 on TiHA surfaces potentiates the burst of gene-mediated osteogenic induction, while it prolongs the osteogenic activity of BMP-6 and surprisingly annihilates the BMP-7 one. These results correlate with FN/BMP affinity for TiHA, since BMP-6>BMP-2>BMP-7. In addition, by analyzing the osteogenic activity in the peri-implant environment, we showed that osteoinductive paracrine effects were significantly decreased upon (FN/BMP-6), as opposed to (FN/BMP-2) coatings. Altogether, our results support the use of FN/BMP-6 to develop a biomimetic microenvironment capable to induce osteogenic activity under physiological conditions, with minimum paracrine signalization. The originality of our paper relies on the first direct comparison of the in vitro osteogenic potential of three osteogenic BMPs (BMP-2, -6 and -7) combined with native human plasma fibronectin delivered in solution or coated by laser transfer onto titanium hydroxyapatite surfaces. We confirm that BMP association with fibronectin enhances the osteogenic activity of BMP-2, -6 and -7, but with essential discrepancies, depending on the BMP member, and in agreement with the affinity of BMPs for fibronectin. Moreover, we bring elements to explain the origin of the BMP-2 medical life-threatening side-effects by analyzing in vitro paracrine effects. Finally, this work supports the alternative use of FN/BMP-6 to induce osteogenic activity under physiological conditions, with minimum side effects. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Matrix stiffness-upregulated LOXL2 promotes fibronectin production, MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation.

PubMed

Wu, Sifan; Zheng, Qiongdan; Xing, Xiaoxia; Dong, Yinying; Wang, Yaohui; You, Yang; Chen, Rongxin; Hu, Chao; Chen, Jie; Gao, Dongmei; Zhao, Yan; Wang, Zhiming; Xue, Tongchun; Ren, Zhenggang; Cui, Jiefeng

2018-05-04

Higher matrix stiffness affects biological behavior of tumor cells, regulates tumor-associated gene/miRNA expression and stemness characteristic, and contributes to tumor invasion and metastasis. However, the linkage between higher matrix stiffness and pre-metastatic niche in hepatocellular carcinoma (HCC) is still largely unknown. We comparatively analyzed the expressions of LOX family members in HCC cells grown on different stiffness substrates, and speculated that the secreted LOXL2 may mediate the linkage between higher matrix stiffness and pre-metastatic niche. Subsequently, we investigated the underlying molecular mechanism by which matrix stiffness induced LOXL2 expression in HCC cells, and explored the effects of LOXL2 on pre-metastatic niche formation, such as BMCs recruitment, fibronectin production, MMPs and CXCL12 expression, cell adhesion, etc. RESULTS: Higher matrix stiffness significantly upregulated LOXL2 expression in HCC cells, and activated JNK/c-JUN signaling pathway. Knockdown of integrin β1 and α5 suppressed LOXL2 expression and reversed the activation of above signaling pathway. Additionally, JNK inhibitor attenuated the expressions of p-JNK, p-c-JUN, c-JUN and LOXL2, and shRNA-c-JUN also decreased LOXL2 expression. CM-LV-LOXL2-OE and rhLOXL2 upregulated MMP9 expression and fibronectin production obviously in lung fibroblasts. Moreover, activation of Akt pathway contributed to LOXL2-induced fibronectin upregulation. LOXL2 in CM as chemoattractant increased motility and invasion of BMCs, implicating a significant role of LOXL2 in BMCs recruitment. Except that, CM-LV-LOXL2-OE as chemoattractant also increased the number of migrated HCC cells, and improved chemokine CXCL12 expression in lung fibroblasts. The number of HCC cells adhered to surface of lung fibroblasts treated with CM-LV-LOXL2-OE was remarkably higher than that of the control cells. These results indicated that the secreted LOXL2 facilitated the motility of HCC cells and strengthened CTCs settlement on the remodeled matrix "soil". Integrin β1/α5/JNK/c-JUN signaling pathway participates in higher matrix stiffness-induced LOXL2 upregulation in HCC cells. The secreted LOXL2 promotes fibronectin production, MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation.

Delineating fibronectin bioadhesive micropatterns by photochemical immobilization of polystyrene and poly(vinylpyrrolidone).

PubMed

Sterner, Olof; Giazzon, Marta; Zürcher, Stefan; Tosatti, Samuele; Liley, Martha; Spencer, Nicholas D

2014-01-01

Bioadhesive micropatterns, capable of laterally confining cells to a 2D lattice, have proven effective in simulating the in vivo tissue environment. They reveal fundamental aspects of the role of adhesion in cell mechanics, proliferation, and differentiation. Here we present an approach based on photochemistry for the fabrication of synthetic polymer micropatterns. Perfluorophenyl azide (PFPA), upon deep-UV exposure, forms a reactive nitrene capable of covalently linking to a molecule that is in close proximity. PFPA has been grafted onto a backbone of poly(allyl amine), which readily forms a self-assembled monolayer on silicon wafers or glass. A film of polystyrene was applied by spin-coating, and by laterally confining the UV exposure through a chromium-on-quartz photomask, monolayers of polymers could be immobilized in circular microdomains. Poly(vinylpyrrolidone) (PVP) was attached to the background to form a barrier to nonspecific protein adsorption and cell adhesion. Micropatterns were characterized with high-lateral-resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS), which confirmed the formation of polystyrene domains within a PVP background. Fluorescence-microscopy adsorption assays with rhodamine-labeled bovine serum albumin demonstrated the nonfouling efficiency of PVP and, combined with TOF-SIMS, allowed for a comprehensive characterization of the pattern geometry. The applicability of the micropatterned platform in single-cell assays was tested by culturing two cell types, WM 239 melanoma cells and SaOs-2 osteoblasts, on micropatterned glass, either with or without backfilling of the patterns with fibronectin. It was demonstrated that the platform was efficient in confining cells to the fibronectin-backfilled micropatterns for at least 48 h. PVP is thus proposed as a viable, highly stable alternative to poly(ethylene glycol) for nonfouling applications. Due to the versatility of the nitrene-insertion reaction, the platform could be extended to other polymer pairs or proteins and the surface chemistry adapted to specific applications.

Actin Cytoskeletal Disruption following Cryopreservation Alters the Biodistribution of Human Mesenchymal Stromal Cells In Vivo

PubMed Central

Chinnadurai, Raghavan; Garcia, Marco A.; Sakurai, Yumiko; Lam, Wilbur A.; Kirk, Allan D.; Galipeau, Jacques; Copland, Ian B.

2014-01-01

Summary Mesenchymal stromal cells have shown clinical promise; however, variations in treatment responses are an ongoing concern. We previously demonstrated that MSCs are functionally stunned after thawing. Here, we investigated whether this cryopreservation/thawing defect also impacts the postinfusion biodistribution properties of MSCs. Under both static and physiologic flow, compared with live MSCs in active culture, MSCs thawed from cryopreservation bound poorly to fibronectin (40% reduction) and human endothelial cells (80% reduction), respectively. This reduction correlated with a reduced cytoskeletal F-actin content in post-thaw MSCs (60% reduction). In vivo, live human MSCs could be detected in murine lung tissues for up to 24 hr, whereas thawed MSCs were undetectable. Similarly, live MSCs whose actin cytoskeleton was chemically disrupted were undetectable at 24 hr postinfusion. Our data suggest that post-thaw cryopreserved MSCs are distinct from live MSCs. This distinction could significantly affect the utility of MSCs as a cellular therapeutic. PMID:25068122

Triclosan Promotes Staphylococcus aureus Nasal Colonization

PubMed Central

Syed, Adnan K.; Ghosh, Sudeshna; Love, Nancy G.; Boles, Blaise R.

2014-01-01

ABSTRACT The biocide triclosan is used in many personal care products, including toothpastes, soaps, clothing, and medical equipment. Consequently, it is present as a contaminant in the environment and has been detected in some human fluids, including serum, urine, and milk. Staphylococcus aureus is an opportunistic pathogen that colonizes the noses and throats of approximately 30% of the population. Colonization with S. aureus is known to be a risk factor for several types of infection. Here we demonstrate that triclosan is commonly found in the nasal secretions of healthy adults and the presence of triclosan trends positively with nasal colonization by S. aureus. We demonstrate that triclosan can promote the binding of S. aureus to host proteins such as collagen, fibronectin, and keratin, as well as inanimate surfaces such as plastic and glass. Lastly, triclosan-exposed rats are more susceptible to nasal colonization with S. aureus. These data reveal a novel factor that influences the ability of S. aureus to bind surfaces and alters S. aureus nasal colonization. PMID:24713325

Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

PubMed

Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

2016-01-01

As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

Transglutaminases factor XIII-A and TG2 regulate resorption, adipogenesis and plasma fibronectin homeostasis in bone and bone marrow

PubMed Central

Mousa, Aisha; Cui, Cui; Song, Aimei; Myneni, Vamsee D; Sun, Huifang; Li, Jin Jin; Murshed, Monzur; Melino, Gerry; Kaartinen, Mari T

2017-01-01

Appropriate bone mass is maintained by bone-forming osteoblast and bone-resorbing osteoclasts. Mesenchymal stem cell (MSC) lineage cells control osteoclastogenesis via expression of RANKL and OPG (receptor activator of nuclear factor κB ligand and osteoprotegerin), which promote and inhibit bone resorption, respectively. Protein crosslinking enzymes transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A) have been linked to activity of myeloid and MSC lineage cells; however, in vivo evidence has been lacking to support their function. In this study, we show in mice that TG2 and FXIII-A control monocyte-macrophage cell differentiation into osteoclasts as well as RANKL production in MSCs and in adipocytes. Long bones of mice lacking TG2 and FXIII-A transglutaminases, show compromised biomechanical properties and trabecular bone loss in axial and appendicular skeleton. This was caused by increased osteoclastogenesis, a cellular phenotype that persists in vitro. The increased potential of TG2 and FXIII-A deficient monocytes to form osteoclasts was reversed by chemical inhibition of TG activity, which revealed the presence of TG1 in osteoclasts and assigned different roles for the TGs as regulators of osteoclastogenesis. TG2- and FXIII-A-deficient mice had normal osteoblast activity, but increased bone marrow adipogenesis, MSCs lacking TG2 and FXIII-A showed high adipogenic potential and significantly increased RANKL expression as well as upregulated TG1 expression. Chemical inhibition of TG activity in the null cells further increased adipogenic potential and RANKL production. Altered differentiation of TG2 and FXIII-A null MSCs was associated with plasma fibronectin (FN) assembly defect in cultures and FN retention in serum and marrow in vivo instead of assembly into bone. Our findings provide new functions for TG2, FXIII-A and TG1 in bone cells and identify them as novel regulators of bone mass, plasma FN homeostasis, RANKL production and myeloid and MSC cell differentiation. PMID:28387755

αMβ2-integrin-intercellular adhesion molecule-1 interactions drive the flow-dependent trafficking of Guillain-Barré syndrome patient derived mononuclear leukocytes at the blood-nerve barrier in vitro

PubMed Central

Yosef, Nejla; Ubogu, Eroboghene E.

2012-01-01

The mechanisms of hematogenous leukocyte trafficking at the human blood-nerve barrier (BNB) are largely unknown. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the pathogenesis of Guillain-Barré syndrome (GBS). We developed a cytokine-activated human in vitro BNB model using primary endoneurial endothelial cells. Endothelial treatment with 10 U/mL tissue necrosis factor-α and 20 U/mL interferon-γ resulted in de novo expression of proinflammatory chemokines CCL2, CXCL9, CXCL11 and CCL20, with increased expression of CXCL2-3, CXCL8 and CXCL10 relative to basal levels. Cytokine treatment induced/ enhanced ICAM-1, E- and P-selectin, vascular cell adhesion molecule-1 and the alternatively spliced pro-adhesive fibronectin variant, fibronectin connecting segment-1 expression in a time-dependent manner, without alterations in junctional adhesion molecule-A expression. Lymphocytes and monocytes from untreated GBS patients express ICAM-1 counterligands, αM- and αL-integrin, with differential regulation of αM-integrin expression compared to healthy controls. Under flow conditions that mimic capillary hemodynamics in vivo, there was a >3-fold increase in total GBS patient and healthy control mononuclear leukocyte adhesion/ migration at the BNB following cytokine treatment relative to the untreated state. Function neutralizing monoclonal antibodies against human αM-integrin (CD11b) and ICAM-1 reduced untreated GBS patient mononuclear leukocyte trafficking at the BNB by 59% and 64.2% respectively. Monoclonal antibodies against αL-integrin (CD11a) and human intravenous immunoglobulin reduced total leukocyte adhesion/migration by 22.8% and 17.6% respectively. This study demonstrates differential regulation of αM-integrin on circulating mononuclear cells in GBS, as well as an important role for αM-integrin-ICAM-1 interactions in pathogenic GBS patient leukocyte trafficking at the human BNB in vitro. PMID:22552879

Restriction fragment length polymorphisms for growth hormone, prolactin, osteonectin, alpha crystallin, gamma crystallin, fibronectin and 21-steroid hydroxylase in cattle.

PubMed

Theilmann, J L; Skow, L C; Baker, J F; Womack, J E

1989-01-01

Genomic DNAs from animals representing six breeds of cattle (Angus, Brahman, Hereford, Holstein, Jersey and Texas Longhorn) were screened with cloned gene probes in a search for restriction fragment length polymorphisms (RFLPs). Eleven RFLPs were identified using seven different probes: growth hormone, prolactin, osteonectin, alpha A-crystallin, gamma crystallin, fibronectin and 21-steroid hydroxylase. The frequencies of the alleles identified by each probe were calculated and compared in a limited sampling of the six bovine breeds. These polymorphisms greatly enhance the pool of immunogenetic, biochemical and molecular markers available in cattle for linkage analysis, testing of parentage, and distinction of breeds.

xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies.

PubMed

Martinez-Serra, Jordi; Gutierrez, Antonio; Muñoz-Capó, Saúl; Navarro-Palou, María; Ros, Teresa; Amat, Juan Carlos; Lopez, Bernardo; Marcus, Toni F; Fueyo, Laura; Suquia, Angela G; Gines, Jordi; Rubio, Francisco; Ramos, Rafael; Besalduch, Joan

2014-01-01

The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562). With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs.

A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER)

PubMed Central

Munfus, Delicia L; Haga, Christopher L; Burrows, Peter D; Cooper, Max D

2007-01-01

Background In mouse the cytokine interleukin-7 (IL-7) is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER). The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR), a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules. PMID:17854505

[Knocking-out extra domain A alternative splice fragment of fibronectin using a clustered regularly interspaced short palindromic repeats/associated proteins 9 system].

PubMed

Yang, Yue; Wang, Haicheng; Xu, Shuyu; Peng, Jing; Jiang, Jiuhui; Li, Cuiying

2015-08-01

To investigate the effect of the fibronectin extra domain A on the aggressiveness of salivary adenoid cystic carcinoma (SACC) cells, via the clustered regularly interspaced short palindromic repeats (CRISPR)/ associated proteins (Cas) system. One sgRNA was designed to target the upstream of the genome sequences of extra domain A(EDA) exon and the downstream. Then the sgRNA was linked into plasmid PX-330 and transfected into SACC-83 cells. PCR and DNA sequence were used to testify the knockout cells, and the monoclones of EDA absent SACC cells were selected (A+C-2, A+C-6, B+C-10). CCK-8 cell proliferation and invasion was then tested in control group and the experimental group. The sgRNA was successfully linked into PX-330 plasmid. Part of adenoid cystic carcinoma cells' SACC-83 genomic EDA exon was knocked out, and the knockdown efficiency was above 70%, but the total amount of fibronectin did not change significantly. Three monoclones of EDA absent SACC- 83 cells were successfully selected with diminished migration and proliferation. The CRISPR/Cas9 system was a simplified system with relatively high knockout efficiency and EDA knockout could inhibiting SACC cell's mobility and invasiveness.

In vivo comparison of biomimetic approaches for tissue regeneration of the scarred vocal fold.

PubMed

Thibeault, Susan L; Klemuk, Sarah A; Smith, Marshall E; Leugers, Cecilia; Prestwich, Glenn

2009-07-01

The objective of this study was to determine if three different biomimetic approaches could facilitate tissue regeneration and improve viscoelastic properties in the scarred vocal fold lamina propria extracellular matrix (ECM). Twenty rabbit vocal folds were biopsied bilaterally; 2 months postinjury rabbits were unilaterally treated with (i) autologous fibroblasts, (ii) a semisynthetic ECM (sECM), or (iii) autologous fibroblasts encapsulated in sECM. Saline was injected as a control into the contralateral fold. Animals were sacrificed 2 months after treatment. Outcomes measured were procollagen, collagen, and fibronectin levels in the lamina propria, and tissue viscosity and elasticity across three frequency decades. All treatment groups demonstrated accelerated proliferation of the ECM. Vocal fold lamina propria treated with autologous fibroblasts were found to have significantly improved viscosity (p = 0.0077) and elasticity (p = 0.0081) compared to saline. This treatment group had significantly elevated fibronectin levels. sECM and autologous fibroblasts/sECM groups had significantly elevated levels of procollagen, collagen, and fibronectin, indicating abundant matrix production as compared to saline with viscoelastic measures that did not differ statistically from controls. The use of autologous fibroblasts led to better restoration of the vocal fold lamina propria biomechanical properties. Optimization of cell-scaffold interactions and subsequent cell behavior is necessary for utilization of scaffold and scaffold-cell approaches.

Laminin and Fibronectin in Cell Adhesion: Enhanced Adhesion of Cells from Regenerating Liver to Laminin

NASA Astrophysics Data System (ADS)

Carlsson, Roland; Engvall, Eva; Freeman, Aaron; Ruoslahti, Erkki

1981-04-01

Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5-6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance of a proper tissue organization during liver regeneration.

Campylobacter jejuni CsrA Regulates Metabolic and Virulence Associated Proteins and Is Necessary for Mouse Colonization.

PubMed

Fields, Joshua A; Li, Jiaqi; Gulbronson, Connor J; Hendrixson, David R; Thompson, Stuart A

2016-01-01

Campylobacter jejuni infection is a leading bacterial cause of gastroenteritis and a common antecedent leading to Gullian-Barré syndrome. Our previous data suggested that the RNA-binding protein CsrA plays an important role in regulating several important phenotypes including motility, biofilm formation, and oxidative stress resistance. In this study, we compared the proteomes of wild type, csrA mutant, and complemented csrA mutant C. jejuni strains in an effort to elucidate the mechanisms by which CsrA affects virulence phenotypes. The putative CsrA regulon was more pronounced at stationary phase (111 regulated proteins) than at mid-log phase (25 regulated proteins). Proteins displaying altered expression in the csrA mutant included diverse metabolic functions, with roles in amino acid metabolism, TCA cycle, acetate metabolism, and various other cell processes, as well as pathogenesis-associated characteristics such as motility, chemotaxis, oxidative stress resistance, and fibronectin binding. The csrA mutant strain also showed altered autoagglutination kinetics when compared to the wild type. CsrA specifically bound the 5' end of flaA mRNA, and we demonstrated that CsrA is a growth-phase dependent repressor of FlaA expression. Finally, the csrA mutant exhibited reduced ability to colonize in a mouse model when in competition with the wild type, further underscoring the role of CsrA in C. jejuni colonization and pathogenesis.

Campylobacter jejuni CsrA Regulates Metabolic and Virulence Associated Proteins and Is Necessary for Mouse Colonization

PubMed Central

Fields, Joshua A.; Li, Jiaqi; Gulbronson, Connor J.; Hendrixson, David R.

2016-01-01

Campylobacter jejuni infection is a leading bacterial cause of gastroenteritis and a common antecedent leading to Gullian-Barré syndrome. Our previous data suggested that the RNA-binding protein CsrA plays an important role in regulating several important phenotypes including motility, biofilm formation, and oxidative stress resistance. In this study, we compared the proteomes of wild type, csrA mutant, and complemented csrA mutant C. jejuni strains in an effort to elucidate the mechanisms by which CsrA affects virulence phenotypes. The putative CsrA regulon was more pronounced at stationary phase (111 regulated proteins) than at mid-log phase (25 regulated proteins). Proteins displaying altered expression in the csrA mutant included diverse metabolic functions, with roles in amino acid metabolism, TCA cycle, acetate metabolism, and various other cell processes, as well as pathogenesis-associated characteristics such as motility, chemotaxis, oxidative stress resistance, and fibronectin binding. The csrA mutant strain also showed altered autoagglutination kinetics when compared to the wild type. CsrA specifically bound the 5’ end of flaA mRNA, and we demonstrated that CsrA is a growth-phase dependent repressor of FlaA expression. Finally, the csrA mutant exhibited reduced ability to colonize in a mouse model when in competition with the wild type, further underscoring the role of CsrA in C. jejuni colonization and pathogenesis. PMID:27257952

The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

PubMed Central

Gu, Changkyu; Park, Soochul

2001-01-01

Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via α5β1- or β3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110γ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110γ. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110γ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110γ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion. PMID:11416136

Nanophase hydroxyapatite coatings for dental and orthopedic applications

NASA Astrophysics Data System (ADS)

Sato, Michiko

In order to improve dental and orthopedic implant performance, the objective of this study was to synthesize nanocrystalline hydroxyapatite (HA) powders to coat metals (specifically, titanium and tantalum). Precipitated HA powders were either sintered in order to produce UltraCaP HA (or microcrystalline size HA) or were treated hydrothermally to produce nanocrystalline HA. Some of the UltraCaP and nanocrystalline HA powders were doped with yttrium (Y) since previous in vitro studies demonstrated that Y-doped HA in bulk improved osteoblast (or bone-forming cell) function over undoped HA. The nanocrystalline HA powders were also mixed with nanophase titania powders because previous studies demonstrated that titania/HA composite coatings increased coating adhesive strength and HA nucleation. These powders were then deposited onto titanium by a novel room-temperature process, called IonTiteT(TM). The results demonstrated that the chemical properties and crystallite size of the original HA powders were maintained in the coatings. More importantly, in vitro studies showed increased osteoblast (bone-forming cell) adhesion on the single phase nanocrystalline HA and nano-titania/HA coatings compared to traditionally used plasma-sprayed HA coatings and uncoated metals. Results further demonstrated greater amounts of calcium deposition by osteoblasts cultured on nanocrystalline HA coatings compared to UltraCaP coatings and conventionally used plasma-sprayed HA coatings. To elucidate mechanisms that influenced osteoblast functions on the HA coatings, the amount of proteins (fibronectin and vitronectin) onto the HA powders and the adsorbed fibronectin conformation were investigated. Exposure of cell integrin binding domains (in fibronectin III10 segments) was greater in fibronectin adsorbed onto 1.2 mole% Y-doped UltraCaP HA coatings compared to nanocrystalline HA coatings tested. However, 1.2 mole% Y-doped UltraCaP HA coatings did not increase mineralization by osteoblasts compared to the nanocrystalline HA coatings. These results suggested that the availability of integrin binding domains in fibronectin did not correlate to enhanced mineralization by osteoblasts on nanocrystalline HA coatings. Lastly, undoped nanocrystalline HA coatings were studied using a well-established rat calvaria in vivo. Histological analysis showed that nanocrystalline HA coated on tantalum scaffolds increased bone and fibrous tissue infiltration into the scaffolds while uncoated and UltraCaP HA coated scaffolds did not after as early as 6 weeks. In summary, these results encourage further studies on nanocrystalline IonTiteTM HA coatings on various metals for orthopedic and dental applications.

The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

PubMed Central

2011-01-01

Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry. Conclusion Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion. PMID:22204307

The Circular RNA Interacts with STAT3, Increasing Its Nuclear Translocation and Wound Repair by Modulating Dnmt3a and miR-17 Function.

PubMed

Yang, Zhen-Guo; Awan, Faryal Mehwish; Du, William W; Zeng, Yan; Lyu, Juanjuan; Wu, De; Gupta, Shaan; Yang, Weining; Yang, Burton B

2017-09-06

Delayed or impaired wound healing is a major health issue worldwide, especially in patients with diabetes and atherosclerosis. Here we show that expression of the circular RNA circ-Amotl1 accelerated healing process in a mouse excisional wound model. Further studies showed that ectopic circ-Amotl1 increased protein levels of Stat3 and Dnmt3a. The increased Dnmt3a then methylated the promoter of microRNA miR-17, decreasing miR-17-5p levels but increasing fibronectin expression. We found that Stat3, similar to Dnmt3a and fibronectin, was a target of miR-17-5p. Decreased miR-17-5p levels would increase expression of fibronectin, Dnmt3a, and Stat3. All of these led to increased cell adhesion, migration, proliferation, survival, and wound repair. Furthermore, we found that circ-Amotl1 not only increased Stat3 expression but also facilitated Stat3 nuclear translocation. Thus, the ectopic expressed circ-Amotl1 and Stat3 were mainly translocated to nucleus. In the presence of circ-Amotl1, Stat3 interacted with Dnmt3a promoter with increased affinity, facilitating Dnmt3a transcription. Ectopic application of circ-Amotl1 accelerating wound repair may shed light on skin wound healing clinically. Copyright © 2017. Published by Elsevier Inc.

The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine

DOE PAGES

Julier, Ziad; Martino, Mikaël M.; de Titta, Alexandre; ...

2015-02-24

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with itsmore » neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. We reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8 + T cell responses in two murine cancer models.« less

Surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains.

PubMed

Chiang, Chunyi; Karuri, Stella W; Kshatriya, Pradnya P; Schwartz, Jeffrey; Schwarzbauer, Jean E; Karuri, Nancy W

2012-01-10

We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.

Influence of aldosterone and salt or ouabain in a10 rat aorta smooth muscle cells.

PubMed

Schwerdt, Gerald; Frisch, Annett; Mildenberger, Sigrid; Hilgenfeld, Tim; Grossmann, Claudia; Gekle, Michael

2012-01-01

It is currently under debate whether aldosterone is able to induce fibrosis or whether it acts only as a cofactor under pathological conditions, e.g. as an elevated salt (NaCl) load. We tested the interaction of 10 nM aldosterone, 15 mM NaCl and 1 μM ouabain using rat aorta smooth muscle cells (A10) with respect to the following parameters: necrosis, apoptosis, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase activity, glutathione (GSH) content, collagen and fibronectin homeostasis and intracellular calcium distribution. Necrosis rates were increased after 48 h of incubation with aldosterone, salt or ouabain and in the combination of aldosterone and salt or ouabain. Apoptosis rates were decreased. A reduced defense capacity against oxidative stress was mirrored in the decreased G6PD activity and GSH content. Collagen III or fibronectin synthesis rates were unchanged, but gelatinase activity was increased resulting in a decreased media collagen III and fibronectin content. Calcium stores were increased by aldosterone in combination with ouabain. Aldosterone and salt per se can lead to cell injury that is aggravated in combination or with cardiotonic steroids. In cooperation with other vascular cells, this can generate a permissive milieu enabling aldosterone or salt to promote more extensive vascular injury. Copyright © 2012 S. Karger AG, Basel.

An anthocyanin rich strawberry extract induces apoptosis and ROS while decreases glycolysis and fibrosis in human uterine leiomyoma cells

PubMed Central

Janjusevic, Milijana; Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y.; Mazzoni, Luca; Greco, Stefania; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Mezzetti, Bruno; Capocasa, Franco; Castellucci, Mario; Battino, Maurizio; Ciarmela, Pasquapina

2017-01-01

Uterine leiomyomas are highly prevalent benign tumors in reproductive aged women. Unfortunately, medical treatments are still limited and no preventive therapies have been developed. In the present study, we investigated the therapeutic effects of strawberry extract on uterine leiomyoma cells. Leiomyoma and myometrial cells were treated with strawberry (cultivar Alba) extract (250 μg/ml) for 48 h to measure apoptosis, reactive oxygen species (ROS), oxidative phosphorylation (OCR, oxygen consumption rate) and glycolysis (ECAR, extracellular acidification rate) as well as fibrosis associated gene and/or protein expression. In leiomyoma cells, strawberry increased the percentage of apoptotic and dead cells. Strawberry significantly increased ROS concentration in leiomyoma cells, while decreased it in myometrial cells. After strawberry treatment, leiomyoma cells showed a significant decreased rate of ECAR, while OCR was unchanged in both myometrial and leiomyoma cells. Strawberry significantly decreased collagen1A1, fibronectin and versican mRNA expression in leiomyoma cells. The reduced protein expression of fibronectin was observed by strawberry extract in leiomyoma cells as well. Furthermore, strawberry was able to reduce activin A induced fibronectin, collagen1A1, and versican as well as activin A and PAI-1 mRNA expression in leiomyoma cells. This study suggests that strawberry can be developed as therapeutic and/or preventive agent for uterine leiomyomas. PMID:28212568

An anthocyanin rich strawberry extract induces apoptosis and ROS while decreases glycolysis and fibrosis in human uterine leiomyoma cells.

PubMed

Islam, Md Soriful; Giampieri, Francesca; Janjusevic, Milijana; Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Mazzoni, Luca; Greco, Stefania; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Mezzetti, Bruno; Capocasa, Franco; Castellucci, Mario; Battino, Maurizio; Ciarmela, Pasquapina

2017-04-04

Uterine leiomyomas are highly prevalent benign tumors in reproductive aged women. Unfortunately, medical treatments are still limited and no preventive therapies have been developed. In the present study, we investigated the therapeutic effects of strawberry extract on uterine leiomyoma cells. Leiomyoma and myometrial cells were treated with strawberry (cultivar Alba) extract (250 μg/ml) for 48 h to measure apoptosis, reactive oxygen species (ROS), oxidative phosphorylation (OCR, oxygen consumption rate) and glycolysis (ECAR, extracellular acidification rate) as well as fibrosis associated gene and/or protein expression. In leiomyoma cells, strawberry increased the percentage of apoptotic and dead cells. Strawberry significantly increased ROS concentration in leiomyoma cells, while decreased it in myometrial cells. After strawberry treatment, leiomyoma cells showed a significant decreased rate of ECAR, while OCR was unchanged in both myometrial and leiomyoma cells. Strawberry significantly decreased collagen1A1, fibronectin and versican mRNA expression in leiomyoma cells. The reduced protein expression of fibronectin was observed by strawberry extract in leiomyoma cells as well. Furthermore, strawberry was able to reduce activin A induced fibronectin, collagen1A1, and versican as well as activin A and PAI-1 mRNA expression in leiomyoma cells. This study suggests that strawberry can be developed as therapeutic and/or preventive agent for uterine leiomyomas.

Focal Adhesion Kinase Regulates Fibroblast Migration via Integrin beta-1 and Plays a Central Role in Fibrosis

PubMed Central

Zhao, Xue-Ke; Cheng, Yiju; Liang Cheng, Ming; Yu, Lei; Mu, Mao; Li, Hong; Liu, Yang; Zhang, Baofang; Yao, Yumei; Guo, Hui; Wang, Rong; Zhang, Quan

2016-01-01

Lung fibrosis is a major medical problem for the aging population worldwide. Fibroblast migration plays an important role in fibrosis. Focal Adhesion Kinase (FAK) senses the extracellular stimuli and initiates signaling cascades that promote cell migration. This study first examined the dose and time responses of FAK activation in human lung fibroblasts treated with platelet derived growth factor BB (PDGF-BB). The data indicate that FAK is directly recruited by integrin β1 and the subsequent FAK activation is required for fibroblast migration on fibronectin. In addition, the study has identified that α5β1 and α4β1 are the major integrins for FAK-mediated fibroblast migration on fibronect. In contrast, integrins αvβ3, αvβ6, and αvβ8 play a minor but distinct role in fibroblast migration on fibronectin. FAK inhibitor significantly reduces PDGF-BB stimulated fibroblast migration. Importantly, FAK inhibitor protects bleomycin-induced lung fibrosis in mice. FAK inhibitor blocks FAK activation and significantly reduces signaling cascade of fibroblast migration in bleomycin-challenged mice. Furthermore, FAK inhibitor decreases lung fibrotic score, collagen accumulation, fibronectin production, and myofibroblast differentiation in in bleomycin-challenged mice. These data demonstrate that FAK mediates fibroblast migration mainly via integrin β1. Furthermore, the findings suggest that targeting FAK signaling is an effective therapeutic strategy against fibrosis. PMID:26763945

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling.

PubMed

Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

2017-01-30

In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

Ultrasonic Stimulation of Mouse Skin Reverses the Healing Delays in Diabetes and Aging by Activation of Rac1.

PubMed

Roper, James A; Williamson, Rosalind C; Bally, Blandine; Cowell, Christopher A M; Brooks, Rebecca; Stephens, Phil; Harrison, Andrew J; Bass, Mark D

2015-11-01

Chronic skin-healing defects are one of the leading challenges to lifelong well-being, affecting 2-5% of populations. Chronic wound formation is linked to age and diabetes and frequently leads to major limb amputation. Here we identify a strategy to reverse fibroblast senescence and improve healing rates. In healthy skin, fibronectin activates Rac1 in fibroblasts, causing migration into the wound bed, and driving wound contraction. We discover that mechanical stimulation of the skin with ultrasound can overturn healing defects by activating a calcium/CamKinaseII/Tiam1/Rac1 pathway that substitutes for fibronectin-dependent signaling and promotes fibroblast migration. Treatment of diabetic and aged mice recruits fibroblasts to the wound bed and reduces healing times by 30%, restoring healing rates to those observed in young, healthy animals. Ultrasound treatment is equally effective in rescuing the healing defects of animals lacking fibronectin receptors, and can be blocked by pharmacological inhibition of the CamKinaseII pathway. Finally, we discover that the migration defects of fibroblasts from human venous leg ulcer patients can be reversed by ultrasound, demonstrating that the approach is applicable to human chronic samples. By demonstrating that this alternative Rac1 pathway can substitute for that normally operating in the skin, we identify future opportunities for management of chronic wounds.

Fibronectin Attachment Protein (FAP) From Bacillus Calmette-Guerin As Targeting Agent For Bladder Tumor Cells

PubMed Central

Coon, Brian G.; Crist, Scott; González-Bonet, Andrés M.; Kim, Hee-Kwon; Sowa, Jennifer; Thompson, David H.; Ratliff, Timothy L.; Aguilar, R. Claudio

2011-01-01

The adjuvant therapy of choice for superficial bladder cancer is the intravesical instillation of live Mycobacterium bovis Bacillus Calmette-Guerin (BCG). In spite of the fact that this therapy is the most effective treatment for superficial bladder cancer, intravesical administration of BCG is associated with high local morbidity and the potential for systemic infection. Therefore, there is a need for the development of safer, less toxic approaches to fight this disease. Since fibronectin attachment protein (FAP) is a key element in BCG retention and targeting to cells, we hypothesize that this protein can be used as targeting agent to deliver cytotoxic cargo for the treatment of bladder tumors. Here we evaluated the ability of bladder tumor cells to bind and endocytose FAP via fibronectin-integrin complexes. We found that microaggregation induced by an anti-FAP polyclonal antibody accelerated FAP uptake by T24 bladder tumor cells. FAP was determined to be internalized via a clathrin-independent, caveolae-dependent mechanism. Further, once within the endosomal compartment, FAP was targeted to the lysosomal compartment with negligible recycling to the plasma membrane. Importantly, we demonstrated that FAP microaggregation and internalization could also be triggered by multivalent Ni2+NTA-bearing liposomes. Overall, our studies validate the use of FAP as a targeting vector and provide the foundation for the design of more effective, less toxic bladder cancer therapeutics. PMID:21901746

Expression, immunogenicity and variation of iron-regulated surface protein A from bovine isolates of Staphylococcus aureus.

PubMed

Misra, Neha; Wines, Tyler F; Knopp, Colton L; McGuire, Mark A; Tinker, Juliette K

2017-05-01

Staphylococcus aureus iron-regulated surface protein A (IsdA) is a fibrinogen and fibronectin adhesin that also contributes to iron sequestration and resistance to innate immunity. IsdA is conserved in human isolates and has been investigated as a human vaccine candidate. Here we report the expression of isdA, the efficacy of anti-IsdA responses and the existence of IsdA sequence variants from bovine Staphylococcus. Clinical staphylococci were obtained from US dairy farms and assayed by PCR for the presence and expression of isdA. isdA-positive species from bovines included S. aureus, S. haemolyticus and S. chromogenes. Immunoassays on bovine milk and serum confirmed the induction and opsonophagocytic activity of anti-IsdA humoral responses. The variable region of isdA was sequenced and protein alignments predicted the presence of two main variants consistent with those from human S. aureus. Mouse antibodies against one IsdA variant reduced staphylococcal binding to fibronectin in vitro in an isotype-dependent manner. Purified IsdA variants bound distinctly to fibronectin and fibrinogen. Our findings demonstrate that variability within the C-terminus of this adhesin affects immune reactivity and binding specificity, but are consistent with the significance of IsdA in bovine disease and relevant for vaccine development. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

NASA Astrophysics Data System (ADS)

Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

2017-01-01

In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

β1 Integrins Mediate Attachment of Mesenchymal Stem Cells to Cartilage Lesions

PubMed Central

Zwolanek, Daniela; Flicker, Magdalena; Kirstätter, Elisabeth; Zaucke, Frank; van Osch, Gerjo J.V.M.; Erben, Reinhold G.

2015-01-01

Abstract Mesenchymal stem cells (MSC) may have great potential for cell-based therapies of osteoarthritis. However, after injection in the joint, only few cells adhere to defective articular cartilage and contribute to cartilage regeneration. Little is known about the molecular mechanisms of MSC attachment to defective articular cartilage. Here, we developed an ex vivo attachment system, using rat osteochondral explants with artificially created full-thickness cartilage defects in combination with genetically labeled MSC isolated from bone marrow of human placental alkaline phosphatase transgenic rats. Binding of MSC to full-thickness cartilage lesions was improved by serum, but not hyaluronic acid, and was dependent on the presence of divalent cations. Additional in vitro tests showed that rat MSC attach, in a divalent cation-dependent manner, to collagen I, collagen II, and fibronectin, but not to collagen XXII or cartilage oligomeric matrix protein (COMP). RGD peptides partially blocked the adhesion of MSC to fibronectin in vitro and to cartilage lesions ex vivo. Furthermore, the attachment of MSC to collagen I and II in vitro and to cartilage lesions ex vivo was almost completely abolished in the presence of a β1 integrin blocking antibody. In conclusion, our data suggest that attachment of MSC to ex vivo full-thickness cartilage lesions is almost entirely β1 integrin-mediated, whereby both RGD- and collagen-binding integrins are involved. These findings suggest a key role of integrins during MSC attachment to defective cartilage and may pave the way for improved MSC-based therapies in the future. PMID:26309781

TGFβ loss activates ADAMTS-1-mediated EGF-dependent invasion in a model of esophageal cell invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le Bras, Grégoire F.; Taylor, Chase; Koumangoye, Rainelli B.

2015-01-01

The TGFβ signaling pathway is essential to epithelial homeostasis and is often inhibited during progression of esophageal squamous cell carcinoma. Recently, an important role for TGFβ signaling has been described in the crosstalk between epithelial and stromal cells regulating squamous tumor cell invasion in mouse models of head-and-neck squamous cell carcinoma (HNSCC). Loss of TGFβ signaling, in either compartment, leads to HNSCC however, the mechanisms involved are not well understood. Using organotypic reconstruct cultures (OTC) to model the interaction between epithelial and stromal cells that occur in dysplastic lesions, we show that loss of TGFβ signaling promotes an invasive phenotypemore » in both fibroblast and epithelial compartments. Employing immortalized esophageal keratinocytes established to reproduce common mutations of esophageal squamous cell carcinoma, we show that treatment of OTC with inhibitors of TGFβ signaling (A83-01 or SB431542) enhances invasion of epithelial cells into a fibroblast-embedded Matrigel/collagen I matrix. Invasion induced by A83-01 is independent of proliferation but relies on protease activity and expression of ADAMTS-1 and can be altered by matrix density. This invasion was associated with increased expression of pro-inflammatory cytokines, IL1 and EGFR ligands HB-EGF and TGFα. Altering EGF signaling prevented or induced epithelial cell invasion in this model. Loss of expression of the TGFβ target gene ROBO1 suggested that chemorepulsion may regulate keratinocyte invasion. Taken together, our data show increased invasion through inhibition of TGFβ signaling altered epithelial-fibroblasts interactions, repressing markers of activated fibroblasts, and altering integrin-fibronectin interactions. These results suggest that inhibition of TGFβ signaling modulates an array of pathways that combined promote multiple aspects of tumor invasion. - Highlights: • Chemical inhibition of TGFβ signaling advances collective invasion of esophageal keratinocytes. • Collective cell invasion is protease dependent and ADAMTS-1 is upregulated. • Inhibition of TGFβ signaling promotes EGF-mediated invasion. • Loss of TGFβ signaling promotes collagen binding and induces alterations in integrin expression. • The TGFβ target ROBO1 is downregulated in cell invasion implying mechanisms of chemorepulsion.« less

The solid state environment orchestrates embryonic development and tissue remodeling

NASA Technical Reports Server (NTRS)

Damsky, C. H.; Moursi, A.; Zhou, Y.; Fisher, S. J.; Globus, R. K.

1997-01-01

Cell interactions with extracellular matrix and with other cells play critical roles in morphogenesis during development and in tissue homeostasis and remodeling throughout life. Extracellular matrix is information-rich, not only because it is comprised of multifunctional structural ligands for cell surface adhesion receptors, but also because it contains peptide signaling factors, and proteinases and their inhibitors. The functions of these groups of molecules are extensively interrelated. In this review, three primary cell culture models are described that focus on adhesion receptors and their roles in complex aspects of morphogenesis and remodeling: the regulation of proteinase expression by fibronectin and integrins in synovial fibroblasts; the regulation of osteoblast differentiation and survival by fibronectin, and the regulation of trophoblast differentiation and invasion by integrins, cadherins and immunoglobulin family adhesion receptors.

Amino acids and peptides. XXXII: A bifunctional poly(ethylene glycol) hybrid of fibronectin-related peptides.

PubMed

Maeda, M; Izuno, Y; Kawasaki, K; Kaneda, Y; Mu, Y; Tsutsumi, Y; Lem, K W; Mayumi, T

1997-12-18

An amino acid type poly(ethylene glycol) (aaPPEG) was prepared and its application to a drug carrier was examined. The peptides, Arg-Gly-Asp (RGD) and Glu-Ile-Leu-Asp-Val (EILDV) which were reported as active fragments of Fibronectin (a cell adhesion protein), were conjugated with aaPEG (molecular weight, 10,000). The hybrid, RGD-aaPEG-EILDV, was prepared by a combination of the solid-phase method and the solution method. Antiadhesive activity of the peptides was not lost by its hybrid formation with the large aaPEG molecule. A mixture of RGD (0.43 mmol) and EILDV (0.43 mmol) did not demonstrate an antiadhesive effect, but the hybrid containing 0.43 mmol of each peptide did exhibit this effect.

Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells.

PubMed

Radwanska, Agata; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Ciais, Delphine; Rekima, Samah; Rupp, Tristan; Sudaka, Anne; Orend, Gertraud; Van Obberghen-Schilling, Ellen

2017-10-06

Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells.

HGF/scatter factor selectively promotes cell invasion by increasing integrin avidity.

PubMed

Trusolino, L; Cavassa, S; Angelini, P; Andó, M; Bertotti, A; Comoglio, P M; Boccaccio, C

2000-08-01

Hepatocyte growth factor/scatter factor (HGF/SF) controls a genetic program known as 'invasive growth', which involves as critical steps cell adhesion, migration, and trespassing of basement membranes. We show here that in MDA-MB-231 carcinoma cells, these steps are elicited by HGF/SF but not by epidermal growth factor (EGF). Neither factor substantially alters the production or activity of extracellular matrix proteases. HGF/SF, but not EGF, selectively promotes cell adhesion on laminins 1 and 5, fibronectin, and vitronectin through a PI3-K-dependent mechanism. Increased adhesion is followed by enhanced invasiveness through isolated matrix proteins as well as through reconstituted basement membranes. Inhibition assays using function-blocking antibodies show that this phenomenon is mediated by multiple integrins including beta1, beta3, beta4, and beta5. HGF/SF triggers clustering of all these integrins at actin-rich adhesive sites and lamellipodia but does not quantitatively modify their membrane expression. These data suggest that HGF/SF promotes cell adhesion and invasiveness by increasing the avidity of integrins for their specific ligands.

Staurosporine and Extracellular Matrix Proteins Mediate the Conversion of Small Cell Lung Carcinoma Cells into a Neuron-Like Phenotype

PubMed Central

Veit, Nadine; Courts, Cornelius; Glassmann, Alexander; Janzen, Viktor; Madea, Burkhard; Reinartz, Markus; Harzen, Anne; Nowak, Michael; Perner, Sven; Winter, Jochen; Probstmeier, Rainer

2014-01-01

Small cell lung carcinomas (SCLCs) represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions. PMID:24586258

Amyloid β-Protein as a Substrate Interacts with Extracellular Matrix to Promote Neurite Outgrowth

NASA Astrophysics Data System (ADS)

Koo, Edward H.; Park, Lisa; Selkoe, Dennis J.

1993-05-01

Progressive deposition of amyloid β-protein (Aβ) in brain parenchyma and blood vessels is a characteristic feature of Alzheimer disease. Recent evidence suggests that addition of solubilized synthetic Aβ to medium may produce toxic or trophic effects on cultured hippocampal neurons. Because soluble Aβ may not accumulate in significant quantities in brain, we asked whether immobilized Aβ peptide as a substrate alters neurite outgrowth from cultured rat peripheral sensory neurons. This paradigm may closely mimic the conditions in Alzheimer disease brain tissue, in which neurites contact insoluble, extracellular aggregates of β-amyloid. We detected no detrimental effects of Aβ substrate on neurite outgrowth. Rather, Aβ in combination with low doses of laminin or fibronectin enhanced neurite out-growth from these neuronal explants. Our results suggest that insoluble Aβ in the cerebral neuropil may serve as a neurite-promoting matrix, perhaps explaining the apparent regenerative response of neurites observed around amyloid plaques in Alzheimer disease. Moreover, in concert with the recent discovery of Aβ production by cultured neurons, our data suggest that Aβ plays a normal physiological role in brain by complexing with the extracellular matrix.

Exposure to diethylhexyl phthalate (DEHP) and monoethylhexyl phthalate (MEHP) promotes the loss of alveolar epithelial phenotype of A549 cells.

PubMed

Rafael-Vázquez, L; García-Trejo, Semiramis; Aztatzi-Aguilar, O G; Bazán-Perkins, B; Quintanilla-Vega, B

2018-05-17

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer that is metabolized to mono(2-ethylhexyl) phthalate (MEHP). Inhalation is an important exposure route for both phthalates, and their effects on lungs include inflammation, alteration of postnatal maturation (alveolarization), enlarged airspaces and cell differentiation changes, suggesting that alveolar epithelial cells-2 (AEC) are targets of phthalates. This study evaluated the cell progression, epithelial and mesenchymal markers, including surfactant secretion in A549 cells (AEC) that were exposed to DEHP (1-100 μM) or MEHP (1-50 μM) for 24-72 h. The results showed an increased cell proliferation at all concentrations of each phthalate at 24 and 48 h. Cell migration showed a concentration-dependent increase at 24 and 48 h of exposure to either phthalate and enlarged structures were seen. Decreased levels of both surfactants (SP-B/SP-C) were observed after the exposure to either phthalate at 48 h, and of SP-C positive cells exposed to MEHP, suggesting a loss of the epithelial phenotype. While a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker fibronectin were observed following exposure to either phthalate. Our results showed that DEHP and MEHP altered the structure and migration of A549 cells and promoted the loss of the epithelial phenotype. Copyright © 2018 Elsevier B.V. All rights reserved.

Aging-induced dysregulation of dicer1-dependent microRNA expression impairs angiogenic capacity of rat cerebromicrovascular endothelial cells.

PubMed

Ungvari, Zoltan; Tucsek, Zsuzsanna; Sosnowska, Danuta; Toth, Peter; Gautam, Tripti; Podlutsky, Andrej; Csiszar, Agnes; Losonczy, Gyorgy; Valcarcel-Ares, M Noa; Sonntag, William E; Csiszar, Anna

2013-08-01

Age-related impairment of angiogenesis is likely to play a central role in cerebromicrovascular rarefaction and development of vascular cognitive impairment, but the underlying mechanisms remain elusive. To test the hypothesis that dysregulation of Dicer1 (ribonuclease III, a key enzyme of the microRNA [miRNA] machinery) impairs endothelial angiogenic capacity in aging, primary cerebromicrovascular endothelial cells (CMVECs) were isolated from young (3 months old) and aged (24 months old) Fischer 344 × Brown Norway rats. We found an age-related downregulation of Dicer1 expression both in CMVECs and in small cerebral vessels isolated from aged rats. In aged CMVECs, Dicer1 expression was increased by treatment with polyethylene glycol-catalase. Compared with young cells, aged CMVECs exhibited altered miRNA expression profile, which was associated with impaired proliferation, adhesion to vitronectin, collagen and fibronectin, cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing technology), and impaired ability to form capillary-like structures. Overexpression of Dicer1 in aged CMVECs partially restored miRNA expression profile and significantly improved angiogenic processes. In young CMVECs, downregulation of Dicer1 (siRNA) resulted in altered miRNA expression profile associated with impaired proliferation, adhesion, migration, and tube formation, mimicking the aging phenotype. Collectively, we found that Dicer1 is essential for normal endothelial angiogenic processes, suggesting that age-related dysregulation of Dicer1-dependent miRNA expression may be a potential mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging.

Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

PubMed

Tang, Bolin; Zhang, Bo; Zhuang, Junjun; Wang, Qi; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

2018-07-01

Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding state of integrin α5β1 with fibronectin induces effective activation of integrin-mediated FAK/ERK signaling pathway to upregulate cellular osteogenic differentiation. This work provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin - Hyaluronic acid cardiac patches.

PubMed

Yang, Ming-Chia; Chi, Nai-Hsin; Chou, Nai-Kuan; Huang, Yi-You; Chung, Tze-Wen; Chang, Yu-Lin; Liu, Hwa-Chang; Shieh, Ming-Jium; Wang, Shoei-Shen

2010-02-01

Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC (rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5-aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9+/-7.1 (%) or 48.4+/-6.0 (%) (n=3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8x10(5)/cell, in fluorescent intensity) while cells with a CD44-blockage treatment markedly diminished the expressions (e.g., 1.1x10(4)/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore, the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44-blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches.

Eosinophils enhance WNT-5a and TGF-β1 genes expression in airway smooth muscle cells and promote their proliferation by increased extracellular matrix proteins production in asthma.

PubMed

Januskevicius, Andrius; Vaitkiene, Simona; Gosens, Reinoud; Janulaityte, Ieva; Hoppenot, Deimante; Sakalauskas, Raimundas; Malakauskas, Kestutis

2016-06-13

Recent studies have suggested that eosinophils may have a direct effect on airway smooth muscle cells (ASMC), causing their proliferation in patients with asthma, but the precise mechanism of the interaction between these cells remains unknown. We propose that changes in Wnt signaling activity and extracellular matrix (ECM) production may help explain these findings. Therefore, the aim of this study was to investigate the effect of eosinophils from asthmatic and non-asthmatic subjects on Wnt-5a, transforming growth factor β1 (TGF-β1), and ECM protein (fibronectin and collagen) gene expression and ASMC proliferation. A total of 18 subjects were involved in the study: 8 steroid-free asthma patients and 10 healthy subjects. Peripheral blood eosinophils were isolated using centrifugation and magnetic separation. An individual co-culture of eosinophils with human ASMC was prepared for each study subject. Adhesion of eosinophils to ASMC (evaluated by assaying eosinophil peroxidase activity) was determined following various incubation periods (30, 45, 60, 120, and 240 min). The expression of Wnt-5a, TGF-β1, and ECM protein genes in ASMC was measured using quantitative real-time polymerase chain reaction (PCR) after 24 h of co-culture. Proliferation of ASMC was measured using the Alamar blue method after 48 h and 72 h of co-culture with eosinophils. Eosinophils from asthmatic subjects demonstrated increased adhesion to ASMC compared with eosinophils from healthy subjects (p < 0.05) in vitro. The expression of Wnt-5a, TGF-β1, collagen, and fibronectin genes in ASMC was significantly higher after 24 h of co-culture with eosinophils from asthmatic subjects, while co-culture of ASMC with eosinophils from healthy subjects increased only TGF-β1 and fibronectin gene expression. ASMC proliferation was augmented after co-culture with eosinophils from asthma patients compared with co-culture with eosinophils from healthy subjects (p < 0.05). Eosinophils enhance Wnt-5a, TGF-β1, fibronectin, and collagen gene expression in ASMC and promote proliferation of these cells in asthma. ClinicalTrials.gov Identifier: NCT02648074 .

Dexamethasone-Mediated Activation of Fibronectin Matrix Assembly Reduces Dispersal of Primary Human Glioblastoma Cells

PubMed Central

Shannon, Stephen; Vaca, Connan; Jia, Dongxuan; Entersz, Ildiko; Schaer, Andrew; Carcione, Jonathan; Weaver, Michael; Avidar, Yoav; Pettit, Ryan; Nair, Mohan; Khan, Atif; Foty, Ramsey A.

2015-01-01

Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM) is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA), increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex), a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively “glues” cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU-486. Our results describe a new role for Dex as a suppressor of GBM dispersal and growth. PMID:26284619

Disruption of integrin-fibronectin complexes by allosteric but not ligand-mimetic inhibitors.

PubMed

Mould, A Paul; Craig, Susan E; Byron, Sarah K; Humphries, Martin J; Jowitt, Thomas A

2014-12-15

Failure of Arg-Gly-Asp (RGD)-based inhibitors to reverse integrin-ligand binding has been reported, but the prevalence of this phenomenon among integrin heterodimers is currently unknown. In the present study we have investigated the interaction of four different RGD-binding integrins (α5β1, αVβ1, αVβ3 and αVβ6) with fibronectin (FN) using surface plasmon resonance. The ability of inhibitors to reverse ligand binding was assessed by their capacity to increase the dissociation rate of pre-formed integrin-FN complexes. For all four receptors we showed that RGD-based inhibitors (such as cilengitide) were completely unable to increase the dissociation rate. Formation of the non-reversible state occurred very rapidly and did not rely on the time-dependent formation of a high-affinity state of the integrin, or the integrin leg regions. In contrast with RGD-based inhibitors, Ca2+ (but not Mg2+) was able to greatly increase the dissociation rate of integrin-FN complexes, with a half-maximal response at ~0.4 mM Ca2+ for αVβ3-FN. The effect of Ca2+ was overcome by co-addition of Mn2+, but not Mg2+. A stimulatory anti-β1 monoclonal antibody (mAb) abrogated the effect of Ca2+ on α5β1-FN complexes; conversely, a function-blocking mAb mimicked the effect of Ca2+. These results imply that Ca2+ acts allosterically, probably through binding to the adjacent metal-ion-dependent adhesion site (ADMIDAS), and that the α1 helix in the β subunit I domain is the key element affected by allosteric modulators. The data suggest an explanation for the limited clinical efficacy of RGD-based integrin antagonists, and we propose that allosteric antagonists could prove to be of greater therapeutic benefit.

Genetics Home Reference: fibronectin glomerulopathy

MedlinePlus

... of tiny blood vessels in the kidneys that filter waste products from blood . The waste products are ... the kidneys, probably as the body attempts to filter it out as waste . Even though there is ...

A new pressure chamber to study the biosynthetic response of articular cartilage to mechanical loading.

PubMed

Steinmeyer, J; Torzilli, P A; Burton-Wurster, N; Lust, G

1993-01-01

A prototype chamber was used to apply a precise cyclic or static load on articular cartilage explants under sterile conditions. A variable pressure, pneumatic controller was constructed to power the chamber's air cylinder, capable of applying, with a porous load platen, loads of up to 10 MPa at cycles ranging from 0 to 10 Hz. Pig articular cartilage explants were maintained successfully in this chamber for 2 days under cyclic mechanical loading of 0.5 Hz, 0.5 MPa. Explants remained sterile, viable and metabolically active. Cartilage responded to this load with a decreased synthesis of fibronectin and a small but statistically significant elevation in proteoglycan content. Similar but less extensive effects on fibronectin synthesis were observed with the small static load (0.016 MPa) inherent in the design of the chamber.

Adhesion of Lactobacillus iners AB-1 to human fibronectin: a key mediator for persistence in the vagina?

PubMed

McMillan, Amy; Macklaim, Jean M; Burton, Jeremy P; Reid, Gregor

2013-07-01

Lactobacillus iners is prominent in the human vagina and is able to persist despite development of bacterial vaginosis and treatment with antibiotics. A probable factor in its persistent survival is its ability to be retained in the vaginal epithelia. Genome sequencing of the strain showed an organism deplete of many metabolic pathways, yet equipped with fibronectin (Fn)-binding adhesins. The objective of the present study was to assess the ability of L iners AB-1 to bind immobilized Fn. Results showed that the organism superiorly bound the protein compared to other species of Lactobacillus and known binders such as Staphylococcus aureus. Treatment of L iners cells by protease rendered its binding abilities to Fn nonfunctional. The findings indicate a mechanism of vaginal persistence for a Lactobacillus species, with implications for reproductive health.

Spray-painted human fibronectin coating as an effective strategy to enhance graft ligamentization of a polyethylene terephthalate artificial ligament.

PubMed

Li, Hong; Chen, Chen; Ge, Yunsheng; Chen, Shiyi

2014-05-01

To enhance graft ligamentization after anterior cruciate ligament (ACL) reconstruction, human fibronectin (FN) was coated on polyethylene terephthalate (PET) ligaments by spray painting. The FN-coated PET ligaments were investigated in vitro using rat mesenchymal stromal cells (MSCs). MSCs cultured on FN-coated grafts resulted in similar cell densities and amounts of proliferating cells with control grafts without coating. The FN-coated group not only gave rise to MSC-derived collagen-like tissues but also enhanced the expression of collagen-I gene. Furthermore, rat ACL reconstruction models were used to evaluate the effect of the FN coating in vivo. The FN coating significantly promoted new ligament tissue regeneration into the graft fibers. In conclusion, sprayed FN coating had a positive effect to enhance graft ligamentization of PET artificial ligament.

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion.

PubMed

Okahashi, Nobuo; Nakata, Masanobu; Sakurai, Atsuo; Terao, Yutaka; Hoshino, Tomonori; Yamaguchi, Masaya; Isoda, Ryutaro; Sumitomo, Tomoko; Nakano, Kazuhiko; Kawabata, Shigetada; Ooshima, Takashi

2010-01-08

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains. Copyright 2009 Elsevier Inc. All rights reserved.

A role for exon sequences in alternative splicing of the human fibronectin gene.

PubMed Central

Mardon, H J; Sebastio, G; Baralle, F E

1987-01-01

Exon EDIIIA of the fibronectin (Fn) gene is alternatively spliced via pathways which either skip or include the whole exon in the messenger RNA (mRNA). We have investigated the role of EDIIIA exon sequences in the human Fn gene in determining alternative splicing of this exon during transient expression of alpha globin/Fn minigene hybrids in HeLa cells. We demonstrate that a DNA sequence of 81bp within the central region of exon EDIIIA is required for alternative splicing during processing of the primary transcript to generate both EDIIIA+ and EDIIIA- mRNA's. Furthermore, alternative splicing of EDIIIA only occurs when this sequence is present in the correct orientation since when it is in antisense orientation splicing always occurs via exon-skipping generating EDIIIA- mRNA. Images PMID:3671064

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

PubMed

Moreno-Cencerrado, Alberto; Iturri, Jagoba; Pecorari, Ilaria; D M Vivanco, Maria; Sbaizero, Orfeo; Toca-Herrera, José L

2017-01-01

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Reinke's edema: investigations on the role of MIB-1 and hepatocyte growth factor.

PubMed

Artico, M; Bronzetti, E; Ionta, B; Bruno, M; Greco, A; Ruoppolo, G; De Virgilio, A; Longo, L; De Vincentiis, M

2010-07-08

Reinke's edema is a benign disease of the human vocal fold, which mainly affects the sub-epithelial layer of the vocal fold. Microscopic observations show a strongly oedematous epithelium with loosened intercellular junctions, a disruption of the extracellular connections between mucosal epithelium and connective tissue, closely adherent to the thyroarytenoid muscle. Thickening of the basal layer of epithelium, known as Reinke's space, high deposition of fibronectin and chronic inflammatory infiltration it is also visible. We analyzed, together with the hepatocyte growth factor (HGF), the expression level of MIB-1 in samples harvested from patients affected by Reinke's edema, in order to define its biological role and consider it as a possible prognostic factor in the follow-up after surgical treatment. We observed a moderate expression of HGF in the lamina propria of the human vocal fold and in the basal membrane of the mucosal epithelium. Our finding suggests that this growth factor acts as an antifibrotic agent in Reinke's space and affects the fibronectin deposition in the lamina propria. MIB-1, on the contrary, showed a weak expression in the basement membrane of the mucosal epithelium and a total absence in the lamina propria deep layer, thus suggesting that only the superficial layer is actively involved in the reparatory process with a high regenerative capacity, together with a high deposition of fibronectin. The latter is necessary for the cellular connections reconstruction, after the inflammatory infiltration.

Reinke's Edema: investigations on the role of MIB-1 and hepatocyte growth factor

PubMed Central

Artico, M.; Bronzetti, E.; Ionta, B.; Bruno, M.; Greco, A.; Ruoppolo, G.; De Virgilio, A.; Longo, L.; De Vincentiis, M.

2010-01-01

Reinke's edema is a benign disease of the human vocal fold, which mainly affects the sub-epithelial layer of the vocal fold. Microscopic observations show a strongly oedematous epithelium with loosened intercellular junctions, a disruption of the extracellular connections between mucosal epithelium and connective tissue, closely adherent to the thyroarytenoid muscle. Thickening of the basal layer of epithelium, known as Reinke's space, high deposition of fibronectin and chronic inflammatory infiltration it is also visible. We analyzed, together with the hepatocyte growth factor (HGF), the expression level of MIB-1 in samples harvested from patients affected by Reinke's edema, in order to define its biological role and consider it as a possible prognostic factor in the follow-up after surgical treatment. We observed a moderate expression of HGF in the lamina propria of the human vocal fold and in the basal membrane of the mucosal epithelium. Our finding suggests that this growth factor acts as an anti - fibrotic agent in Reinke's space and affects the fibronectin deposition in the lamina propria. MIB-1, on the contrary, showed a weak expression in the basement membrane of the mucosal epithelium and a total absence in the lamina propria deep layer, thus suggesting that only the superficial layer is actively involved in the reparatory process with a high regenerative capacity, together with a high deposition of fibronectin. The latter is necessary for the cellular connections reconstruction, after the inflammatory infiltration. PMID:20819770

Liarozole inhibits transforming growth factor-β3–mediated extracellular matrix formation in human three-dimensional leiomyoma cultures

PubMed Central

Levy, Gary; Malik, Minnie; Britten, Joy; Gilden, Melissa; Segars, James; Catherino, William H.

2014-01-01

Objective To investigate the impact of liarozole on transforming growth factor-β3 (TGF-β3) expression, TGF-β3 controlled profibrotic cytokines, and extracellular matrix formation in a three-dimensional (3D) leiomyoma model system. Design Molecular and immunohistochemical analysis in a cell line evaluated in a three-dimensional culture. Setting Laboratory study. Patient(s) None. Intervention(s) Treatment of leiomyoma and myometrial cells with liarozole and TGF-β3 in a three-dimensional culture system. Main Outcome Measure(s) Quantitative real-time reverse-transcriptase polymerase chain reaction and Western blotting to assess fold gene and protein expression of TGF-β3 and TGF-β3 regulated fibrotic cytokines: collagen 1A1 (COL1A1), fibronectin, and versican before and after treatment with liarozole, and confirmatory immunohistochemical stains of treated three-dimensional cultures. Result(s) Both TGF-β3 gene and protein expression were elevated in leiomyoma cells compared with myometrium in two-dimensional and 3D cultures. Treatment with liarozole decreased TGF-β3 gene and protein expression. Extracellular matrix components versican, COL1A1, and fibronectin were also decreased by liarozole treatment in 3D cultures. Treatment of 3D cultures with TGF-β3 increased gene expression and protein production of COL1A1, fibronectin, and versican. Conclusion(s) Liarozole decreased TGF-β3 and TGF-β3–mediated extracellular matrix expression in a 3D uterine leiomyoma culture system. PMID:24825427

GROWTH INHIBITORY ACTIONS OF PROTHROMBIN ON NORMAL HEPATOCYTES

PubMed Central

Carr, Brian I.; Kar, Siddhartha; Wang, Meifang; Wang, Ziqiu

2007-01-01

Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-γ-carboxy-prothrombin (DCP), the function of which is unknown. We considered that prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin, and DNA synthesis and cytoskeleton were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH) concomitant with a peak of DNA synthesis by the hepatocyte at 24 h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24 h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP. PMID:17490900

High-Throughput Ligand Discovery Reveals a Sitewise Gradient of Diversity in Broadly Evolved Hydrophilic Fibronectin Domains

PubMed Central

Woldring, Daniel R.; Holec, Patrick V.; Zhou, Hong; Hackel, Benjamin J.

2015-01-01

Discovering new binding function via a combinatorial library in small protein scaffolds requires balance between appropriate mutations to introduce favorable intermolecular interactions while maintaining intramolecular integrity. Sitewise constraints exist in a non-spatial gradient from diverse to conserved in evolved antibody repertoires; yet non-antibody scaffolds generally do not implement this strategy in combinatorial libraries. Despite the fact that biased amino acid distributions, typically elevated in tyrosine, serine, and glycine, have gained wider use in synthetic scaffolds, these distributions are still predominantly applied uniformly to diversified sites. While select sites in fibronectin domains and DARPins have shown benefit from sitewise designs, they have not been deeply evaluated. Inspired by this disparity between diversity distributions in natural libraries and synthetic scaffold libraries, we hypothesized that binders resulting from discovery and evolution would exhibit a non-spatial, sitewise gradient of amino acid diversity. To identify sitewise diversities consistent with efficient evolution in the context of a hydrophilic fibronectin domain, >105 binders to six targets were evolved and sequenced. Evolutionarily favorable amino acid distributions at 25 sites reveal Shannon entropies (range: 0.3–3.9; median: 2.1; standard deviation: 1.1) supporting the diversity gradient hypothesis. Sitewise constraints in evolved sequences are consistent with complementarity, stability, and consensus biases. Implementation of sitewise constrained diversity enables direct selection of nanomolar affinity binders validating an efficient strategy to balance inter- and intra-molecular interaction demands at each site. PMID:26383268

Reticuloendothelial hyperphagocytosis occurs in streptozotocin-diabetic rats. Studies with colloidal carbon, albumin microaggregates, and soluble fibrin monomers.

PubMed

Cornell, R P

1982-02-01

In contrast to previous studies of diabetic humans and animals, which reported unchanged or depressed function, reticuloendothelial system (RES) hyperphagocytosis of colloidal carbon, 125I-albumin microaggregates, and 125I-fibrin monomers were observed in rats as early as 14 days after the induction of diabetes with streptozotocin (STZ). The fact that enhanced phagocytosis by RE macrophages was prevented by chronic insulin replacement therapy indicates that the diabetic internal environment of hyperglycemia and hypoinsulinemia was perhaps responsible for the observed changes. Experiments involving organ localization of intravenously administered particles, perfusion of isolated livers, and microscopic examination of the liver all suggested that increased Kupffer cell activity was the primary event in RES hyperphagocytosis by STZ-diabetic rats. Both hypertrophy and hyperplasia of Kupffer cells were apparent in livers of STZ-diabetic animals as evidenced by photomicrographs and hepatic cell quantification. Plasma fibronectin, which binds fibrin monomers to RE macrophages before phagocytosis, was significantly decreased in the circulation of STZ-diabetic rats, but the level of cell-associated fibronectin was not measured. Renal localization of urea-soluble 125I-fibrin monomers exceeded splenic and pulmonary uptake in normal control rats and was enhanced in animals with STZ-diabetes. Changes in fibronectin levels, fibrin monomer localization, and Kupffer cell size and numbers in experimental diabetes in rats may have implications for the pathogenesis of vascular disease involving phagocytic mesangial and foam cells in diabetic humans.

Selective fibronectin adsorption against albumin and enhanced stem cell attachment on helium atmospheric pressure glow discharge treated titanium

NASA Astrophysics Data System (ADS)

Han, Inho; Vagaska, Barbora; Joo Park, Bong; Lee, Mi Hee; Jin Lee, Seung; Park, Jong-Chul

2011-06-01

Successful tissue integration of implanted medical devices depends on appropriate initial cellular response. In this study, the effect of helium atmospheric pressure glow discharge (He-APGD) treatment of titanium on selective protein adsorption and the initial attachment processes and focal adhesion formation of osteoprogenitor cells and stem cells were examined. Titanium disks were treated in a self-designed He-APGD system. Initial attachment of MC3T3-E1 mouse pre-osteoblasts and human mesenchymal stem cells (MSCs) was evaluated by MTT assay and plasma membrane staining followed by morphometric analysis. Fibronectin adsorption was investigated by Enzyme-Linked ImmunoSorbant Assay. MSCs cell attachment to treated and non-treated titanium disks coated with different proteins was verified also in serum-free culture. Organization of actin cytoskeleton and focal adhesions was evaluated microscopically. He-APGD treatment effectively modified the titanium surfaces by creating a super-hydrophilic surface, which promoted selectively higher adsorption of fibronectin, a protein of critical importance for cell/biomaterial interaction. In two different types of cells, the He-APGD treatment enhanced the number of attaching cells as well as their attachment area. Moreover, cells had higher organization of actin cytoskeleton and focal adhesions. Faster acceptance of the material by the progenitor cells in the early phases of tissue integration after the implantation may significantly reduce the overall healing time; therefore, titanium treatment with He-APGD seems to be an effective method of surface modification of titanium for improving its tissue inductive properties.

Neuropeptides, via specific receptors, regulate T cell adhesion to fibronectin.

PubMed

Levite, M; Cahalon, L; Hershkoviz, R; Steinman, L; Lider, O

1998-01-15

The ability of T cells to adhere to and interact with components of the blood vessel walls and the extracellular matrix is essential for their extravasation and migration into inflamed sites. We have found that the beta1 integrin-mediated adhesion of resting human T cells to fibronectin, a major glycoprotein component of the extracellular matrix, is induced by physiologic concentrations of three neuropeptides: calcitonin gene-related protein (CGRP), neuropeptide Y, and somatostatin; each acts via its own specific receptor on the T cell membrane. In contrast, substance P (SP), which coexists with CGRP in the majority of peripheral endings of sensory nerves, including those innervating the lymphoid organs, blocks T cell adhesion to fibronectin when induced by CGRP, neuropeptide Y, somatostatin, macrophage inflammatory protein-1beta, and PMA. Inhibition of T cell adhesion was obtained both by the intact SP peptide and by its 1-4 N-terminal and its 4-11, 5-11, and 6-11 C-terminal fragments, used at similar nanomolar concentrations. The inhibitory effects of the parent SP peptide and its fragments were abrogated by an SP NK-1 receptor antagonist, suggesting they all act through the same SP NK-1 receptor. These findings suggest that neuropeptides, by activating their specific T cell-expressed receptors, can provide the T cells with both positive (proadhesive) and negative (antiadhesive) signals and thereby regulate their function. Thus, neuropeptides may influence diverse physiologic processes involving integrins, including leukocyte-mediated migration and inflammation.

Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with α4β1 integrins

PubMed Central

Vogelezang, Mariette; Forster, Ulrike B; Han, Jaewon; Ginsberg, Mark H; ffrench-Constant, Charles

2007-01-01

Background The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for α4β1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the α4 integrin cytoplasmic domain in neurite outgrowth. Results Expression of α4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type α4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal α4-mediated migration in leucocytes requires spatial regulation of α4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this α4(S988D), which mimics phosphorylated α4, did not promote neurite outgrowth. However, α4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable α4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin. Conclusion We establish the importance of the α4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of α4 for extension of neuronal growth cones and migration of non-neural cells. PMID:17603879

Which Factors Contribute to False-Positive, False-Negative, and Invalid Results in Fetal Fibronectin Testing in Women with Symptoms of Preterm Labor?

PubMed

Bruijn, Merel M C; Hermans, Frederik J R; Vis, Jolande Y; Wilms, Femke F; Oudijk, Martijn A; Kwee, Anneke; Porath, Martina M; Oei, Guid; Scheepers, Hubertina C J; Spaanderman, Marc E A; Bloemenkamp, Kitty W M; Haak, Monique C; Bolte, Antoinette C; Vandenbussche, Frank P H A; Woiski, Mallory D; Bax, Caroline J; Cornette, Jérôme M J; Duvekot, Johannes J; Bijvank, Bas W A N I J; van Eyck, Jim; Franssen, Maureen T M; Sollie, Krystyna M; van der Post, Joris A M; Bossuyt, Patrick M M; Kok, Marjolein; Mol, Ben W J; van Baaren, Gert-Jan

2017-02-01

Objective  We assessed the influence of external factors on false-positive, false-negative, and invalid fibronectin results in the prediction of spontaneous delivery within 7 days. Methods  We studied symptomatic women between 24 and 34 weeks' gestational age. We performed uni- and multivariable logistic regression to estimate the effect of external factors (vaginal soap, digital examination, transvaginal sonography, sexual intercourse, vaginal bleeding) on the risk of false-positive, false-negative, and invalid results, using spontaneous delivery within 7 days as the outcome. Results  Out of 708 women, 237 (33%) had a false-positive result; none of the factors showed a significant association. Vaginal bleeding increased the proportion of positive fetal fibronectin (fFN) results, but was significantly associated with a lower risk of false-positive test results (odds ratio [OR], 0.22; 95% confidence intervals [CI], 0.12-0.39). Ten women (1%) had a false-negative result. None of the investigated factors was significantly associated with a significantly higher risk of false-negative results. Twenty-one tests (3%) were invalid; only vaginal bleeding showed a significant association (OR, 4.5; 95% CI, 1.7-12). Conclusion  The effect of external factors on the performance of qualitative fFN testing is limited, with vaginal bleeding as the only factor that reduces its validity. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Bcr-Abl induces abnormal cytoskeleton remodeling, beta1 integrin clustering and increased cell adhesion to fibronectin through the Abl interactor 1 pathway.

PubMed

Li, Yingzhu; Clough, Nancy; Sun, Xiaolin; Yu, Weidong; Abbott, Brian L; Hogan, Christopher J; Dai, Zonghan

2007-04-15

Hematopoietic cells isolated from patients with Bcr-Abl-positive leukemia exhibit multiple abnormalities of cytoskeletal and integrin function. These abnormalities are thought to play a role in the pathogenesis of leukemia; however, the molecular events leading to these abnormalities are not fully understood. We show here that the Abi1 pathway is required for Bcr-Abl to stimulate actin cytoskeleton remodeling, integrin clustering and cell adhesion. Expression of Bcr-Abl induces tyrosine phosphorylation of Abi1. This is accompanied by a subcellular translocation of Abi1/WAVE2 to a site adjacent to membrane, where an F-actin-enriched structure containing the adhesion molecules such as beta1-integrin, paxillin and vinculin is assembled. Bcr-Abl-induced membrane translocation of Abi1/WAVE2 requires direct interaction between Abi1 and Bcr-Abl, but is independent of the phosphoinositide 3-kinase pathway. Formation of the F-actin-rich complex correlates with an increased cell adhesion to fibronectin. More importantly, disruption of the interaction between Bcr-Abl and Abi1 by mutations either in Bcr-Abl or Abi1 not only abolished tyrosine phosphorylation of Abi1 and membrane translocation of Abi1/WAVE2, but also inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, integrin clustering and cell adhesion to fibronectin. Together, these data define Abi1/WAVE2 as a downstream pathway that contributes to Bcr-Abl-induced abnormalities of cytoskeletal and integrin function.

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features.

PubMed

Karvonen, Henna M; Lehtonen, Siri T; Sormunen, Raija T; Harju, Terttu H; Lappi-Blanco, Elisa; Bloigu, Risto S; Kaarteenaho, Riitta L

2012-09-01

The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.

Study protocol: quantitative fibronectin to help decision-making in women with symptoms of preterm labour (QUIDS) part 2, UK Prospective Cohort Study

PubMed Central

Wotherspoon, Lisa M; Boyd, Kathleen Anne; Morris, Rachel K; Jackson, Lesley; Chandiramani, Manju; David, Anna L; Khalil, Asma; Shennan, Andrew; Hodgetts Morton, Victoria; Lavender, Tina; Khan, Khalid; Harper-Clarke, Susan; Mol, Ben; Riley, Richard D; Norrie, John; Norman, Jane

2018-01-01

Introduction The aim of the QUIDS study is to develop a decision support tool for the management of women with symptoms and signs of preterm labour, based on a validated prognostic model using quantitative fetal fibronectin (fFN) concentration, in combination with clinical risk factors. Methods and analysis The study will evaluate the Rapid fFN 10Q System (Hologic, Marlborough, Massachusetts, USA) which quantifies fFN in a vaginal swab. In QUIDS part 2, we will perform a prospective cohort study in at least eight UK consultant-led maternity units, in women with symptoms of preterm labour at 22+0 to 34+6 weeks gestation to externally validate a prognostic model developed in QUIDS part 1. The effects of quantitative fFN on anxiety will be assessed, and acceptability of the test and prognostic model will be evaluated in a subgroup of women and clinicians (n=30). The sample size is 1600 women (with estimated 96–192 events of preterm delivery within 7 days of testing). Clinicians will be informed of the qualitative fFN result (positive/negative) but be blinded to quantitative fFN result. Research midwives will collect outcome data from the maternal and neonatal clinical records. The final validated prognostic model will be presented as a mobile or web-based application. Ethics and dissemination The study is funded by the National Institute of Healthcare Research Health Technology Assessment (HTA 14/32/01). It has been approved by the West of Scotland Research Ethics Committee (16/WS/0068). Version Protocol V.2, Date 1 November 2016. Trial registration number ISRCTN41598423 and CPMS: 31277. PMID:29674373

Cardiomyocytes In Vitro Adhesion Is Actively Influenced by Biomimetic Synthetic Peptides for Cardiac Tissue Engineering

PubMed Central

Huerta-Cantillo, Rocio; Comisso, Marina; Danesin, Roberta; Ghezzo, Francesca; Naso, Filippo; Gastaldello, Alessandra; Schittullo, Eleonora; Buratto, Edward; Spina, Michele; Gerosa, Gino; Dettin, Monica

2012-01-01

Scaffolds for tissue engineering must be designed to direct desired events such as cell attachment, growth, and differentiation. The incorporation of extracellular matrix-derived peptides into biomaterials has been proposed to mimic biochemical signals. In this study, three synthetic fragments of fibronectin, vitronectin, and stromal-derived factor-1 were investigated for the first time as potential adhesive sequences for cardiomyocytes (CMs) compared to smooth muscle cells. CMs are responsive to all peptides to differing degrees, demonstrating the existence of diverse adhesion mechanisms. The pretreatment of nontissue culture well surfaces with the (Arginine-Glycine-Aspartic Acid) RGD sequence anticipated the appearance of CMs' contractility compared to the control (fibronectin-coated well) and doubled the length of cell viability. Future prospects are the inclusion of these sequences into biomaterial formulation with the improvement in cell adhesion that could play an important role in cell retention during dynamic cell seeding. PMID:22011064

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Jiachao; Zhou, Yufan; Hua, Xin

We demonstrate in situ chemical imaging of protein biomolecules in the aqueous solution using System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane forming the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectra, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that fibronectin film in water has more distinct and strongermore » water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment.« less

Pediatric Origins of Nephrolithiasis-Associated Atherosclerosis.

PubMed

Kusumi, Kirsten; Smith, Sally; Barr-Beare, Evan; Saxena, Vijay; Schober, Megan S; Moore-Clingenpeel, Melissa; Schwaderer, Andrew L

2015-11-01

To determine if nephrolithiasis-associated atherosclerosis has pediatric origins and to consider possible association between kidney stones and atherosclerosis-related proteins. We matched children aged 12-17 years with kidney stones and without kidney stones. Carotid artery intima-media thickness (cIMT) was measured by ultrasound. Participants' urine was investigated by enzyme-linked immunosorbent assay for the atherosclerosis-related proteins fibronectin 1, macrophage scavenger receptor 1, osteopontin, and vascular cell adhesion molecule 1 levels, and normalized to urine creatinine levels. Subjects with nephrolithiasis had higher cIMT in the right common carotid artery and overall mean measurement. Urine osteopontin and fibronectin 1 were significant predictors of cIMT. We have provided initial preliminary evidence that nephrolithiasis-associated atherosclerosis has pediatric origins and performed studies that begin to identify potential reasons for the association of nephrolithiasis and vascular disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Studying Cancer Stem Cell Dynamics on PDMS Surfaces for Microfluidics Device Design

PubMed Central

Zhang, Weijia; Choi, Dong Soon; Nguyen, Yen H.; Chang, Jenny; Qin, Lidong

2013-01-01

This systematic study clarified a few interfacial aspects of cancer cell phenotypes on polydimethylsiloxane (PDMS) substrates and indicated that the cell phenotypic equilibrium greatly responds to cell-to-surface interactions. We demonstrated that coatings of fibronectin, bovine serum albumin (BSA), or collagen with or without oxygen-plasma treatments of the PDMS surfaces dramatically impacted the phenotypic equilibrium of breast cancer stem cells, while the variations of the PDMS elastic stiffness had much less such effects. Our results showed that the surface coatings of collagen and fibronectin on PDMS maintained breast cancer cell phenotypes to be nearly identical to the cultures on commercial polystyrene Petri dishes. The surface coating of BSA provided a weak cell-substrate adhesion that stimulated the increase in stem-cell-like subpopulation. Our observations may potentially guide surface modification approaches to obtain specific cell phenotypes. PMID:23900274

Radiolabeled Escherichia coli heat-stable enterotoxin analogs for in vivo imaging of colorectal cancer

NASA Astrophysics Data System (ADS)

Giblin, M. F.; Sieckman, G. L.; Owen, N. K.; Hoffman, T. J.; Forte, L. R.; Volkert, W. A.

2005-12-01

The human Escherichia coli heat-stable enterotoxin (STh, amino acid sequence N1SSNYCCELCCNPACTGCY19) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. In the current study, two STh analogs were synthesized and evaluated in vitro and in vivo. Both analogs shared identical 6-19 core sequences, and had N-terminal pendant DOTA moieties. The analogs differed in the identity of a 6 amino acid peptide sequence intervening between DOTA and the 6-19 core. In one analog, the peptide was an RGD-containing sequence found in human fibronectin (GRGDSP), while in the other this peptide sequence was randomly scrambled (GRDSGP). The results indicated that the presence of the human fibronectin sequence in the hybrid peptide did not affect tumor localization in vivo.

Matrix control of pancreatic cancer: New insights into fibronectin signaling.

PubMed

Topalovski, Mary; Brekken, Rolf A

2016-10-10

Pancreatic ductal adenocarcinoma (PDA) is a highly metastatic disease that resists most current therapies. A defining characteristic of PDA is an intense fibrotic response that promotes tumor cell invasion and chemoresistance. Efforts to understand the complex relationship between the tumor and its extracellular network and to therapeutically perturb tumor-stroma interactions are ongoing. Fibronectin (FN), a provisional matrix protein abundant in PDA stroma but not normal tissues, supports metastatic spread and chemoresistance of this deadly disease. FN also supports angiogenesis, which is required for even hypovascular tumors such as PDA to develop and progress. Targeting components of the tumor stroma, such as FN, can effectively reduce tumor growth and spread while also enhancing delivery of chemotherapy. Here, we review the molecular mechanisms by which FN drives angiogenesis, metastasis and chemoresistance in PDA. In light of these new findings, we also discuss therapeutic strategies to inhibit FN signaling. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Osteograft, plastic material for regenerative medicine

NASA Astrophysics Data System (ADS)

Zaidman, A. M.; Korel, A. V.; Shevchenko, A. I.; Shchelkunova, E. I.; Sherman, K. M.; Predein, Yu. A.; Kosareva, O. S.

2016-08-01

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14-30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissue specificity of the developed osteograft.

Quantitative fibronectin to help decision-making in women with symptoms of preterm labour (QUIDS) part 1: Individual participant data meta-analysis and health economic analysis

PubMed Central

Wotherspoon, Lisa M; Boyd, Kathleen A; Morris, Rachel K; Jackson, Lesley; Chandiramani, Manju; David, Anna L; Khalil, Asma; Shennan, Andrew; Hodgetts Morton, Victoria; Lavender, Tina; Khan, Khalid; Harper-Clarke, Susan; Mol, Ben W; Riley, Richard D; Norrie, John; Norman, Jane E

2018-01-01

Introduction The aim of the QUIDS study is to develop a decision support tool for the management of women with symptoms and signs of preterm labour, based on a validated prognostic model using quantitative fetal fibronectin (qfFN) concentration, in combination with clinical risk factors. Methods and analysis The study will evaluate the Rapid fFN 10Q System (Hologic, Marlborough, Massachusetts) which quantifies fFN in a vaginal swab. In part 1 of the study, we will develop and internally validate a prognostic model using an individual participant data (IPD) meta-analysis of existing studies containing women with symptoms of preterm labour alongside fFN measurements and pregnancy outcome. An economic analysis will be undertaken to assess potential cost-effectiveness of the qfFN prognostic model. The primary endpoint will be the ability of the prognostic model to rule out spontaneous preterm birth within 7 days. Six eligible studies were identified by systematic review of the literature and five agreed to provide their IPD (n=5 studies, 1783 women and 139 events of preterm delivery within 7 days of testing). Ethics and dissemination The study is funded by the National Institute of Healthcare Research Health Technology Assessment (HTA 14/32/01). It has been approved by the West of Scotland Research Ethics Committee (16/WS/0068). PROSPERO registration number CRD42015027590. Version Protocol version 2, date 1 November 2016. PMID:29627817

Use of the Uteroglobin Platform for the Expression of a Bivalent Antibody against Oncofetal Fibronectin in Escherichia coli

PubMed Central

Ventura, Elisa; Riondato, Mattia; Sambuceti, Gianmario; Salis, Annalisa; Damonte, Gianluca; Cordazzo, Cinzia; Besir, Hüseyin; Pistoia, Vito; Zardi, Luciano

2013-01-01

Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646–26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50–70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli. PMID:24367567

Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

NASA Technical Reports Server (NTRS)

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

2000-01-01

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

Effects of extracellular matrix proteins on macrophage differentiation, growth, and function: comparison of liquid and agar culture systems

NASA Technical Reports Server (NTRS)

Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Both spaceflight and skeletal unloading suppress the haematopoietic differentiation of macrophages (Sonnenfeld et al., Aviat. Space Environ. Med., 61:648-653, 1990; Armstrong et al., J. Appl. Physiol., 75:2734-2739, 1993). The mechanism behind this reduction in haematopoiesis has yet to be elucidated. However, changes in bone marrow extracellular matrix (ECM) may be involved. To further understand the role of ECM products in macrophage differentiation, we have performed experiments evaluating the effects of fibronectin, laminin, collagen type I, and collagen type IV on macrophage development and function. Bone marrow-derived macrophages cultured on four different ECM substrates in liquid culture medium showed less growth than those cultured on plastic. Significant morphological differences were seen on each of the substrates used. Phenotypically and functionally, as measured by class II major histocompatibility molecule (MHCII) expression, MAC-2 expression, and the secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), these macrophages were similar. In contrast, bone marrow-derived macrophages cultured in suspension, using agar, showed no difference in growth when exposed to ECM proteins. However, IL-6 and TNF-alpha secretion was affected by fibronectin, laminin, collagen type I, and collagen type IV in a concentration-dependent manner. We conclude that the ECM products fibronectin, laminin, collagen type I, and collagen type IV have profound effects on macrophage development and function. Additionally, we suggest that an ECM-supplemented agar culture system provides an environment more analogous to in vivo bone marrow than does a traditional liquid culture system.

Fibronectin matrix-mediated cohesion suppresses invasion of prostate cancer cells.

PubMed

Jia, Dongxuan; Entersz, Ildiko; Butler, Christine; Foty, Ramsey A

2012-03-20

Invasion is an important early step in the metastatic cascade and is the primary cause of death of prostate cancer patients. In order to invade, cells must detach from the primary tumor. Cell-cell and cell-ECM interactions are important regulators of cohesion--a property previously demonstrated to mediate cell detachment and invasion. The studies reported here propose a novel role for α5β1 integrin--the principle mediator of fibronectin matrix assembly (FNMA)--as an invasion suppressor of prostate cancer cells. Using a combination of biophysical and cell biological methods, and well-characterized prostate cancer cell lines of varying invasiveness, we explore the relationship between cohesion, invasiveness, and FNMA. We show that cohesion is inversely proportional to invasive capacity. We also show that more invasive cells express lower levels of α5β1 integrin and lack the capacity for FNMA. Cells were generated to over-express either wild-type α5 integrin or an integrin in which the cytoplasmic domain of α5 was replaced with that of α2. The α2 construct does not promote FNMA. We show that only wild-type α5 integrin promotes aggregate compaction, increases cohesion, and reduces invasion of the more aggressive cells, and that these effects can be blocked by the 70-kDa fibronectin fragment. We propose that restoring capacity for FNMA in deficient cells can increase tumor intercellular cohesion to a point that significantly reduces cell detachment and subsequent invasion. In prostate cancer, this could be of therapeutic benefit by blocking an early key step in the metastatic cascade.

Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb Protein Switches Its Activity from Auto-aggregation to Biofilm Formation.

PubMed

Miljkovic, Marija; Bertani, Iris; Fira, Djordje; Jovcic, Branko; Novovic, Katarina; Venturi, Vittorio; Kojic, Milan

2016-01-01

AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix, we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III, and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation.

High-throughput investigation of endothelial-to-mesenchymal transformation (EndMT) with combinatorial cellular microarrays.

PubMed

Wang, Zongjie; Calpe, Blaise; Zerdani, Jalil; Lee, Youngsang; Oh, Jonghyun; Bae, Hojae; Khademhosseini, Ali; Kim, Keekyoung

2016-07-01

In the developing heart, a specific subset of endocardium undergoes an endothelial-to-mesenchymal transformation (EndMT) thus forming nascent valve leaflets. Extracellular matrix (ECM) proteins and growth factors (GFs) play important roles in regulating EndMT but the combinatorial effect of GFs with ECM proteins is less well understood. Here we use microscale engineering techniques to create single, binary, and tertiary component microenvironments to investigate the combinatorial effects of ECM proteins and GFs on the attachment and transformation of adult ovine mitral valve endothelial cells to a mesenchymal phenotype. With the combinatorial microenvironment microarrays, we utilized 60 different combinations of ECM proteins (Fibronectin, Collagen I, II, IV, Laminin) and GFs (TGF-β1, bFGF, VEGF) and were able to identify new microenvironmental conditions capable of modulating EndMT in MVECs. Experimental results indicated that TGF-β1 significantly upregulated the EndMT while either bFGF or VEGF downregulated EndMT process markedly. Also, ECM proteins could influence both the attachment of MVECs and the response of MVECs to GFs. In terms of attachment, fibronectin is significantly better for the adhesion of MVECs among the five tested proteins. Overall collagen IV and fibronectin appeared to play important roles in promoting EndMT process. Great consistency between macroscale and microarrayed experiments and present studies demonstrates that high-throughput cellular microarrays are a promising approach to study the regulation of EndMT in valvular endothelium. Biotechnol. Bioeng. 2016;113: 1403-1412. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Mesenchymal stem cells protect against obstruction-induced renal fibrosis by decreasing STAT3 activation and STAT3-dependent MMP-9 production

PubMed Central

Matsui, Futoshi; Babitz, Stephen A.; Rhee, Audrey; Hile, Karen L.; Zhang, Hongji

2017-01-01

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mesenchymal stem cell (MSC)-induced renoprotection during renal fibrosis remains unknown. We hypothesized that MSCs protect against obstruction-induced renal fibrosis by downregulating STAT3 activation and STAT3-induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 106/rat) immediately before sham operation or induction of unilateral ureteral obstruction (UUO). The kidneys were harvested after 4 wk and analyzed for collagen I and III gene expression, collagen deposition (Masson’s trichrome), fibronectin, α-smooth muscle actin, active STAT3 (p-STAT3), MMP-9, and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) expression. In a separate arm, the STAT3 inhibitor S3I-201 (10 mg/kg) vs. vehicle was administered to rats intraperitoneally just after induction of UUO and daily for 14 days thereafter. The kidneys were harvested after 2 wk and analyzed for p-STAT3 and MMP-9 expression, and collagen and fibronectin deposition. Renal obstruction induced a significant increase in collagen, fibronectin, α-SMA, p-STAT3, MMP-9, and TIMP-1 expression while exogenously administered MSCs significantly reduced these indicators of obstruction-induced renal fibrosis. STAT3 inhibition with S3I-201 significantly reduced obstruction-induced MMP-9 expression and tubulointerstitial fibrosis. These results demonstrate that MSCs protect against obstruction-induced renal fibrosis, in part, by decreasing STAT3 activation and STAT3-dependent MMP-9 production. PMID:27760767

Alpha lipoic acid selectively inhibits proliferation and adhesion to fibronectin of v-H-ras-transformed 3Y1 cells.

PubMed

Yamasaki, Masao; Iwase, Masahiro; Kawano, Kazuo; Sakakibara, Yoichi; Suiko, Masahito; Nishiyama, Kazuo

2012-05-01

Here, we focused on the effects of racemic α-lipoic acid on proliferation and adhesion properties of 3Y1 rat fibroblasts and the v-H-ras-transformed derivative, HR-3Y1-2 cells. Racemic α-lipoic acid inhibited proliferation of HR-3Y1-2 but not 3Y1 cells at 0.3 and 1.0 mM. R-(+)-α-lipoic acid also inhibited proliferation of HR-3Y1-2 cells equivalent to that of racemic α-lipoic acid. In addition, racemic α-lipoic acid decreased intracellular reactive oxygen species levels in HR-3Y1 cells but not 3Y1 cells. Next, we evaluated the effects of racemic α-lipoic acid on cell adhesion to fibronectin. The results indicated that racemic α-lipoic acid decreased adhesive ability of HR-3Y1-2 cells to fibronectin-coated plates. As blocking antibody experiment revealed that β1-integrin plays a key role in cell adhesion in this experimental system, the effects of racemic α-lipoic acid on the expression of β1-integrin were examined. The results indicated that racemic α-lipoic acid selectively downregulated the expression of cell surface β1-integrin expression in HR-3Y1-2 cells. Intriguingly, exogenous hydrogen peroxide upregulated cell surface β1-integrin expression in 3Y1 cells. Taken together, these data suggest that reduction of intracellular reactive oxygen species levels by α-lipoic acid could be an effective means of ameliorating abnormal growth and adhesive properties in v-H-ras transformed cells.

Tensile forces drive a reversible fibroblast-to-myofibroblast transition during tissue growth in engineered clefts

PubMed Central

Kollmannsberger, Philip; Bidan, Cécile M.; Dunlop, John W. C.; Fratzl, Peter; Vogel, Viola

2018-01-01

Myofibroblasts orchestrate wound healing processes, and if they remain activated, they drive disease progression such as fibrosis and cancer. Besides growth factor signaling, the local extracellular matrix (ECM) and its mechanical properties are central regulators of these processes. It remains unknown whether transforming growth factor–β (TGF-β) and tensile forces work synergistically in up-regulating the transition of fibroblasts into myofibroblasts and whether myofibroblasts undergo apoptosis or become deactivated by other means once tissue homeostasis is reached. We used three-dimensional microtissues grown in vitro from fibroblasts in macroscopically engineered clefts for several weeks and found that fibroblasts transitioned into myofibroblasts at the highly tensed growth front as the microtissue progressively closed the cleft, in analogy to closing a wound site. Proliferation was up-regulated at the growth front, and new highly stretched fibronectin fibers were deposited, as revealed by fibronectin fluorescence resonance energy transfer probes. As the tissue was growing, the ECM underneath matured into a collagen-rich tissue containing mostly fibroblasts instead of myofibroblasts, and the fibronectin fibers were under reduced tension. This correlated with a progressive rounding of cells from the growth front inward, with decreased α–smooth muscle actin expression, YAP nuclear translocation, and cell proliferation. Together, this suggests that the myofibroblast phenotype is stabilized at the growth front by tensile forces, even in the absence of endogenously supplemented TGF-β, and reverts into a quiescent fibroblast phenotype already 10 μm behind the growth front, thus giving rise to a myofibroblast-to-fibroblast transition. This is the hallmark of reaching prohealing homeostasis. PMID:29349300

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

PubMed

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

2015-01-01

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

Time-dependent changes in extra-domain A-fibronectin concentration and relative amounts of fibronectin-fibrin complexes in plasma of patients with peripheral arterial disease after endovascular revascularisation.

PubMed

Pupek, Małgorzata; Krzyżanowska-Gołąb, Dorota; Kotschy, Daniel; Witkiewicz, Wojciech; Kwiatkowska, Wiesława; Kotschy, Maria; Kątnik-Prastowska, Iwona

2018-03-13

Fibronectin (FN) may be involved in time- and stage-dependent and inter-related controlled processes of inflammation, coagulation, and wound healing accompanying peripheral arterial disease (PAD). In the present study, FN and FN-containing extra-domain A (EDA-FN), macromolecular FN-fibrin complexes, and FN monomer were analysed in the plasma of 142 PAD patients, including 37 patients with restenosis, for 37 months after revascularisation. FN concentration increased significantly in the plasma of PAD patients within 7 to 12 months after revascularisation, whereas the high concentration of EDA-FN was maintained up to 24 months, significantly higher in the group 7 to 12 months after revascularisation with recurrence of stenosis and lower in the PAD groups 1 to 3 months and 4 to 6 months after revascularisation with comorbid diabetes and ulceration, respectively. The relative amounts of FN-fibrin complexes up to 1600 kDa and FN monomer were significantly higher, within intervals of 4 to 24 months and 4 to 6 months after revascularisation, respectively. Moreover, the relative amounts of 750 to 1600 kDa FN-fibrin complexes within 13 to 24 months after revascularisation were higher in comparison with those in the group without restenosis. In conclusion, high levels of EDA-FN and FN-fibrin complexes could have potential diagnostic value in the management of PAD patients after revascularisation, predicting restenosis risk. © 2018 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Nitric oxide donors rescue diabetic nephropathy through oxidative-stress-and nitrosative-stress-mediated Wnt signaling pathways

PubMed Central

Hsu, Yung-Chien; Lee, Pei-Hsien; Lei, Chen-Chou; Ho, Cheng; Shih, Ya-Hsueh; Lin, Chun-Liang

2015-01-01

Aims/Introduction The role of the renal nitric oxide (NO) system in the pathophysiology of diabetic nephropathy constitutes a very challenging and fertile field for future investigation. The purpose of the present study was to investigate whether NO donors can attenuate diabetic renal fibrosis and apoptosis through modulating oxidative-and nitrosative-stress, and Wnt signaling using in vivo diabetic models. Materials and Methods Diabetic rat was induced by a single intraperitoneal injection of streptozotocin. Rats in each group were intraperitoneally given 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (1 U/kg/day) and vehicle for 28 and 56 consecutive days. Expression of the oxidative-and nitrosative-stress, and Wnt signaling components were examined in kidneys from diabetic animals by quantitative reverse transcription polymerase chain reaction, western blot analysis and immunohistochemical staining. Results NO donor treatment significantly reduced the ratio of kidney weight to bodyweight and proteinuria. This treatment also significantly restored the suppressive effect of diabetes on urinary NO2 + NO3 levels. Immunohistochemistry showed that NO donor treatment significantly reduced transforming growth factor (TGF)-β1, fibronectin, cleaved caspase-3 and triphosphate-biotin nick end-labeling expression in the glomeruli of diabetic rats. We found that diabetes promoted 8-hydroxy-2′-deoxyguanosine, and peroxynitrite expression coincided with reduced endothelial NO synthase expression in glomeruli. Interestingly, NO donor treatment completely removed oxidative stress and nitrosative stress, and restored endothelial NO synthase expression in diabetic renal glomeruli. Immunohistomorphometry results showed that NO donor treatment significantly restored suppressed Wnt5a expression and β-catenin immunoreactivities in glomeruli. Based on laser-captured microdissection for quantitative reverse transcription polymerase chain reaction, diabetes significantly increased TGF-β1, and fibronectin expression coincided with depressed Wnt5a expression. NO donor treatment reduced TGF-β1, fibronectin activation, and the suppressing effect of diabetes on Wnt5a and β-catenin expression in renal glomeruli. Conclusions NO donor treatment alleviates extracellular matrix accumulation and apoptosis in diabetic nephropathy in vivo by not only preventing the diabetes-mediated oxidative and nitrostative stress, but also restoring downregulation of endothelial NO synthase expression and Wnt/β-catenin signaling. These findings suggest that modulation of NO is a viable alternative strategy for rescuing diabetic renal injury. PMID:25621130

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers

PubMed Central

De, Pradip; Carlson, Jennifer H.; Wu, Hui; Marcus, Adam; Leyland-Jones, Brian; Dey, Nandini

2016-01-01

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronection-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP. PMID:27281609

Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension

NASA Technical Reports Server (NTRS)

Wang, N.; Ingber, D. E.

1994-01-01

We have investigated how extracellular matrix (ECM) alters the mechanical properties of the cytoskeleton (CSK). Mechanical stresses were applied to integrin receptors on the apical surfaces of adherent endothelial cells using RGD-coated ferromagnetic microbeads (5.5-microns diameter) in conjunction with a magnetic twisting device. Increasing the number of basal cell-ECM contacts by raising the fibronectin (FN) coating density from 10 to 500 ng/cm2 promoted cell spreading by fivefold and increased CSK stiffness, apparent viscosity, and permanent deformation all by more than twofold, as measured in response to maximal stress (40 dyne/cm2). When the applied stress was increased from 7 to 40 dyne/cm2, the stiffness and apparent viscosity of the CSK increased in parallel, although cell shape, ECM contacts, nor permanent deformation was altered. Application of the same stresses over a lower number ECM contacts using smaller beads (1.4-microns diameter) resulted in decreased CSK stiffness and apparent viscosity, confirming that this technique probes into the depth of the CSK and not just the cortical membrane. When magnetic measurements were carried out using cells whose membranes were disrupted and ATP stores depleted using saponin, CSK stiffness and apparent viscosity were found to rise by approximately 20%, whereas permanent deformation decreased by more than half. Addition of ATP (250 microM) under conditions that promote CSK tension generation in membrane-permeabilized cells resulted in decreases in CSK stiffness and apparent viscosity that could be detected within 2 min after ATP addition, before any measurable change in cell size.(ABSTRACT TRUNCATED AT 250 WORDS).

15-LO/15-HETE mediated vascular adventitia fibrosis via p38 MAPK-dependent TGF-β.

PubMed

Zhang, Li; Li, Yumei; Chen, Minggang; Su, Xiaojie; Yi, Dan; Lu, Ping; Zhu, Daling

2014-02-01

15-Lipoxygenase/15-hydroxyeicosatetraenoic acid (15-LO/15-HETE) is known to modulate pulmonary vascular medial hypertrophy and intimal endothelial cells migration and angiogenesis after hypoxia. However, it is unclear whether 15-HETE affects the adventitia of the pulmonary arterial wall. We performed immunohistochemistry, adventitia fibrosis, pulmonary artery fibroblasts phenotype and extracellular matrix (ECM) deposition to determine the role of 15-HETE in hypoxia-induced pulmonary vascular adventitia remodeling. Our studies showed that O2 deprivation induced adventitia hypertrophy of pulmonary arteries with ECM accumulation in both humans with pulmonary arterial hypertension and hypoxic rats. Hypoxia induced 15-LO expression in adventitia. With the inhibitor, NDGA depressed the hypoxia induced ECM deposition and 15-LO production in hypoxic rats. Hypoxia up-regulated the expression of α-SMA, type-Ia collagen and fibronectin in cultured fibroblasts, which seemed to be due to the increased 15-LO/15-HETE. Exogenous 15-HETE mediated the ECM and phenotypic alterations of the fibroblasts as well. The 15-LO/15-HETE induced adventitia fibrosis and fibroblasts phenotypic alterations depended on signaling of the transforming growth factor-β1 (TGF-β1)/Smad2/3 pathway. P38 mitogen-activated protein kinase (p38 MAPKs) was likely to mediate 15-LO induced TGF-β1 and Smad2/3 activation after hypoxia. The results suggest that adventitia fibrosis is an important event in the hypoxia induced pulmonary arterial remodeling, which relies on 15-LO/15-HETE induced p38 MAPK-dependent TGF-β1/Smad2/3 intracellular signaling systems. © 2013 Wiley Periodicals, Inc.

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells.

PubMed

Silva, Mariana C C; de Paula, Cláudia A A; Ferreira, Joana G; Paredes-Gamero, Edgar J; Vaz, Angela M S F; Sampaio, Misako U; Correia, Maria Tereza S; Oliva, Maria Luiza V

2014-07-01

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity. MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting. BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α1, α6 and β1 integrin subunit expression, and increases α5 subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21. BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells. Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Temporal response of canine flexor tendon to limb suspension

PubMed Central

Thoreson, Andrew R.; Cha, Stephen S.; Zhao, Chunfeng; An, Kai-Nan; Amadio, Peter C.

2010-01-01

Tendon disuse, or stress deprivation, frequently accompanies clinical disorders and treatments, yet the metabolism of tendons subject to stress deprivation has rarely been investigated systematically. The effects of stress deprivation on canine flexor tendon were investigated in this study. One adult canine forepaw was suspended for 21 or 42 days. Control forepaws were collected from dogs that had no intervention on their limbs and paws. The expression of collagen I and III was not significantly altered in the tendons disused for 21 days but was significantly decreased at 42 days (P < 0.03). The expression of collagen II, aggrecan, decorin, and fibronectin was significantly decreased in the tendons in the suspended limbs at 21 days (P < 0.002) and further reduced at 42 days. With stress deprivation, the expression of matrix metalloproteinase 2 (MMP2) was significantly increased (P < 0.004) at 21 and 42 days. The expression of MMP3 was significantly decreased at 21 and 42 days (P < 0.03). The expression of MMP13 was not altered with stress deprivation at 21 and 42 days. The expression of MMP14 was significantly increased at 21 days (P = 0.0015) and returned to the control level at 42 days. Tissue inhibitor of metalloproteinase 1 (TIMP1) expression was decreased after the limbs were suspended for 42 days (P = 0.0043), but not 21 days. However, TIMP2 expression was not significantly different from control at 21 or 42 days. Furthermore, the cross-sectional area of the stress-deprived tendons at 42 days was decreased compared with the control group (P < 0.01). The intervention method in this study did not result in any alteration of stiffness of the tendon. Our study demonstrated that stress deprivation decreases the anabolic process and increases the catabolic process of extracellular matrix in flexor tendon. PMID:20947711

Snail1 transcription factor controls telomere transcription and integrity

PubMed Central

Mazzolini, Rocco; Gonzàlez, Núria; Garcia-Garijo, Andrea; Millanes-Romero, Alba; Peiró, Sandra; Smith, Susan

2018-01-01

Abstract Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFβ-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes. PMID:29059385

Inhibition of extracellular matrix mediated TGF-β signalling suppresses endometrial cancer metastasis

PubMed Central

Sahoo, Subhransu S.; Quah, Min Yuan; Nielsen, Sarah; Atkins, Joshua; Au, Gough G.; Cairns, Murray J.; Nahar, Pravin; Lombard, Janine M.; Tanwar, Pradeep S.

2017-01-01

Although aggressive invasion and distant metastases are an important cause of morbidity and mortality in patients with endometrial cancer (EC), the requisite events determining this propensity are currently unknown. Using organotypic three-dimensional culture of endometrial cancer cell lines, we demonstrated anti-correlated TGF-β signalling gene expression patterns that arise among extracellular matrix (ECM)-attached cells. TGF-β pathway seemed to be active in EC cells forming non-glandular colonies in 3D-matrix but weaker in glandular colonies. Functionally we found that out of several ECM proteins, fibronectin relatively promotes Smad phosphorylation suggesting a potential role in regulating TGF-β signalling in non-glandular colonies. Importantly, alteration of TGF-β pathway induced EMT and MET in both type of colonies through slug protein. The results exemplify a crucial role of TGF-β pathway during EC metastasis in human patients and inhibition of the pathway in a murine model impaired tumour cell invasion and metastasis depicting an attractive target for therapeutic intervention of malignant tumour progression. These findings provide key insights into the role of ECM-derived TGF-β signalling to promote endometrial cancer metastasis and offer an avenue for therapeutic targeting of microenvironment derived signals along with tumour cells. PMID:29069715

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation*

PubMed Central

Bernard, Karen; Logsdon, Naomi J.; Ravi, Saranya; Xie, Na; Persons, Benjamin P.; Rangarajan, Sunad; Zmijewski, Jaroslaw W.; Mitra, Kasturi; Liu, Gang; Darley-Usmar, Victor M.; Thannickal, Victor J.

2015-01-01

Contraction is crucial in maintaining the differentiated phenotype of myofibroblasts. Contraction is an energy-dependent mechanism that relies on the production of ATP by mitochondria and/or glycolysis. Although the role of mitochondrial biogenesis in the adaptive responses of skeletal muscle to exercise is well appreciated, mechanisms governing energetic adaptation of myofibroblasts are not well understood. Our study demonstrates induction of mitochondrial biogenesis and aerobic glycolysis in response to the differentiation-inducing factor transforming growth factor β1 (TGF-β1). This metabolic reprogramming is linked to the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Inhibition of p38 MAPK decreased accumulation of active peroxisome proliferator-activated receptor γ coactivator 1α in the nucleus and altered the translocation of mitochondrial transcription factor A to the mitochondria. Genetic or pharmacologic approaches that block mitochondrial biogenesis or glycolysis resulted in decreased contraction and reduced expression of TGF-β1-induced α-smooth muscle actin and collagen α-2(I) but not of fibronectin or collagen α-1(I). These data indicate a critical role for TGF-β1-induced metabolic reprogramming in regulating myofibroblast-specific contractile signaling and support the concept of integrating bioenergetics with cellular differentiation. PMID:26318453

Modeling continuum of epithelial mesenchymal transition plasticity.

PubMed

Mandal, Mousumi; Ghosh, Biswajoy; Anura, Anji; Mitra, Pabitra; Pathak, Tanmaya; Chatterjee, Jyotirmoy

2016-02-01

Living systems respond to ambient pathophysiological changes by altering their phenotype, a phenomenon called 'phenotypic plasticity'. This program contains information about adaptive biological dynamism. Epithelial-mesenchymal transition (EMT) is one such process found to be crucial in development, wound healing, and cancer wherein the epithelial cells with restricted migratory potential develop motile functions by acquiring mesenchymal characteristics. In the present study, phase contrast microscopy images of EMT induced HaCaT cells were acquired at 24 h intervals for 96 h. The expression study of relevant pivotal molecules viz. F-actin, vimentin, fibronectin and N-cadherin was carried out to confirm the EMT process. Cells were intuitively categorized into five distinct morphological phenotypes. A population of 500 cells for each temporal point was selected to quantify their frequency of occurrence. The plastic interplay of cell phenotypes from the observations was described as a Markovian process. A model was formulated empirically using simple linear algebra, to depict the possible mechanisms of cellular transformation among the five phenotypes. This work employed qualitative, semi-quantitative and quantitative tools towards illustration and establishment of the EMT continuum. Thus, it provides a newer perspective to understand the embedded plasticity across the EMT spectrum.

Protein degradation following treatment with hydrogen peroxide.

PubMed Central

Fligiel, S. E.; Lee, E. C.; McCoy, J. P.; Johnson, K. J.; Varani, J.

1984-01-01

Pretreatment of hemoglobin with 50-5000 nmol hydrogen peroxide (H2O2) increased its susceptibility to proteolysis by a number of purified enzymes, including trypsin, chymotrypsin, elastase, and plasmin, and by the neutral protease of rat peritoneal leukocytes. Pretreatment of the protein substrate with catalase-inactivated H2O2 had no effect. Separation of the proteolytic fragments by G-75 Sephadex gel filtration indicated no apparent differences in the size distribution of the fragments produced by treatment with the H2O2/proteolytic enzyme combination as compared with enzyme treatment alone. A partially purified preparation of rat glomerular basement membrane was also treated with proteolytic enzyme alone or in combination with H2O2. As with the hemoglobin, pretreatment of the glomerular basement membrane with H2O2 increased its susceptibility to subsequent proteolytic attack. In addition, treatment of a basement membrane glycoprotein, fibronectin, with H2O2 also increased its sensitivity to subsequent proteolysis. These results suggest that in addition to their other proinflammatory activities, oxygen-derived metabolites may contribute to tissue destruction by altering the susceptibility of proteins to hydrolytic enzymes. Images Figure 1 PMID:6375392

Changes in p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization.

PubMed

Alexandrova, A; Ivanov, A; Chumakov, P; Kopnin, B; Vasiliev, J

2000-11-23

Effects of p53 expression on cell morphology and motility were studied using the derivatives of p53-null 10(1) mouse fibroblasts with tetracycline-regulated expression of exogenous human p53. Induction of p53 expression was accompanied by significant decrease in extracellular matrix (fibronectin) and reduction of matrix fibrils, diminution of the number and size of focal contacts, decrease of cell areas, establishment of more elongated cell shape and alterations of actin cytoskeleton (actin bundles became thinner, their number and size decreased). Expression of His175 and Gln22/ Ser23 p53 mutants caused no such effects. To study the influence of p53 expression on cell motility we used wound technique and videomicroscopy observation of single living cells. It was found that induction of p53 expression led to increase of lamellar activity of cell edge. However, in spite of enhanced lamellar activity p53-expressing cells migrated to shorter distance and filled the narrow wound in longer time as compared with their p53-null counterparts. Possible mechanisms of the influence of p53 expression on cell morphology and motility are discussed.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)-Cell Interaction and the Resultant Bioeffects at the Single-cell Level.

PubMed

Li, Fenfang; Yuan, Fang; Sankin, Georgy; Yang, Chen; Zhong, Pei

2017-01-10

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

[Contemporary methods for preterm labor diagnostics].

PubMed

Kolev, N; Kovachev, E; Ivanov, S; Kornovski, Y; Tsvetkov, K; Angelova, M; Tsonev, A; Ismail, E

2013-01-01

Authors track current trends in preterm labor diagnostics. The emphasis is laid on biochemical tests for examination of fibronectin (fFN) and insulin-like growth factor-binding protein (IGFBP-1) in cervical and vaginal secretions, as well as ultrasound assessment of cervical length.

Disentangling the multifactorial contributions of fibronectin, collagen and cyclic strain on MMP expression and extracellular matrix remodeling by fibroblasts.

PubMed

Zhang, Yang; Lin, Zhe; Foolen, Jasper; Schoen, Ingmar; Santoro, Alberto; Zenobi-Wong, Marcy; Vogel, Viola

2014-11-01

Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was thus to gain insight into the ECM-driven functional regulation of human foreskin fibroblasts (HFFs) being either anchored to a fibronectin (Fn) or to a collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. While the cells reoriented in response to the onset of uniaxial cyclic strain, cells assembled exogenously added Fn with a preferential Fn-fiber alignment along their new orientation. Exposure of HFFs to exogenous Fn resulted in an increase in matrix metalloproteinase (MMP) expression levels, i.e. MMP-15 (RT-qPCR), and MMP-9 activity (zymography), while subsequent exposure to collagen slightly reduced MMP-15 expression and MMP-9 activity compared to Fn-exposure alone. Cyclic strain upregulated Fn fibrillogenesis and actin stress fiber formation, but had comparatively little effect on MMP activity. We thus propose that the appearance of collagen might start to steer HFFs towards homeostasis, as it decreased both MMP secretion and the tension of Fn matrix fibrils as assessed by Fluorescence Resonance Energy Transfer. These results suggest that HFFs might have a high ECM remodeling or repair capacity in contact with Fn alone (early event), which is reduced in the presence of Col1 (later event), thereby down-tuning HFF activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis. Copyright © 2014. Published by Elsevier B.V.

Urinary fibronectin levels in patients treated with intravesical bacillus Calmette-Guérin for superficial bladder cancer.

PubMed

Danişman, A; Bulut, K; Kukul, E; Ozen, I; Sevük, M

2000-01-01

Intravesical bacillus Calmette-Guérin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma (TCC) of the bladder, but the precise mechanism of action of BCG remains poorly understood. Fibronectin (FN), an important component of the extracellular matrix, has been found to play a role in BCG therapy. Some studies have shown that the soluble form of FN can compete efficiently with the matrix form of binding to the specific receptors on the bacteria and could consequently diminish the effect of BCG treatment. To evaluate a possible correlation between the urinary levels of FN and the efficacy of BCG therapy, we determined prospectively the urinary FN levels in 38 patients with TCC of the bladder and in 25 control subjects without malignancy matched for age and sex. All TCC patients were treated with transurethral tumor resection plus 6 weekly intravesical BCG instillations. After an average follow-up of 30 months, 8 patients (21.1%) had recurrent tumors, while 30 (78.9%) were free of tumor after intravesical BCG therapy. Urinary levels of FN in cancer patients have been shown to be significantly higher than controls (p < 0.001). These elevated levels were not decreased significantly after the operation (p > 0. 05). It was also found that the mean urinary FN levels were not statistically significant between patients with recurrence and complete remission. The data suggest that BCG-bladder tumor cell binding is not influenced by soluble fibronectin and urinary FN may not be a ideal marker for selecting patients to BCG therapy. Copyright 2000 S. Karger AG, Basel

Adhesion mechanisms in embryogenesis and in cancer invasion and metastasis.

PubMed

Thiery, J P; Boyer, B; Tucker, G; Gavrilovic, J; Valles, A M

1988-01-01

Cell-substratum and cell-cell adhesion mechanisms contribute to the development of animal form. The adhesive status of embryonic cells has been analysed during epithelial-mesenchymal cell interconversion and in cell migrations. Clear-cut examples of the modulation of cell adhesion molecules (CAMs) have been described at critical periods of morphogenesis. In chick embryos the three primary CAMs (N-CAM. L-CAM and N-cadherin) present early in embryogenesis are expressed later in a defined pattern during morphogenesis and histogenesis. The axial mesoderm derived from gastrulating cells expresses increasing amounts of N-cadherin and N-CAM. During metamerization these two adhesion molecules become abundant at somitic cell surfaces. Both CAMs are functional in an in vitro aggregation assay; however, the calcium-dependent adhesion molecule N-cadherin is more sensitive to perturbation by specific antibodies. Neural crest cells which separate from the neural epithelium lose their primary CAMs in a defined time-sequence. Adhesion to fibronectins via specific surface receptors becomes a predominant interaction during the migratory process, while some primary and secondary CAMs are expressed de novo during the ontogeny of the peripheral nervous system. In vitro, different fibronectin functional domains have been identified in the attachment, spreading and migration of neural crest cells. The fibronectin receptors which transduce the adhesive signals play a key role in the control of cell movement. All these results have prompted us to examine whether similar mechanisms operate in carcinoma cell invasion and metastasis. In vitro, rat bladder transitional carcinoma cells convert reversibly into invasive mesenchymal cells. A rapid modulation of adhesive properties is found during the epithelial-mesenchymal carcinoma cell interconversion. The different model systems analysed demonstrate that a limited repertoire of adhesion molecules, expressed in a well-defined spatiotemporal pattern, is involved in tissue formation and in key processes of tumour spread.

Gonadotropin-releasing hormone analogues inhibit leiomyoma extracellular matrix despite presence of gonadal hormones.

PubMed

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

2016-01-01

To determine the effect of GnRH analogues (GnRH-a) leuprolide acetate (LA) and cetrorelix acetate on gonadal hormone-regulated expression of extracellular matrix in uterine leiomyoma three-dimensional (3D) cultures. Laboratory study. University research laboratory. Women undergoing hysterectomy for symptomatic leiomyomas. The 3D cell cultures, protein analysis, Western blot, immunohistochemistry. Expression of extracellular matrix proteins, collagen 1, fibronectin, and versican in leiomyoma cells 3D cultures exposed to E2, P, LA, cetrorelix acetate, and combinations for 24- and 72-hour time points. The 3D leiomyoma cultures exposed to E2 for 24 hours demonstrated an increased expression of collagen-1 and fibronectin, which was maintained for up to 72 hours, a time point at which versican was up-regulated significantly. Although P up-regulated collagen-1 protein (1.29 ± 0.04) within 24 hours of exposure, significant increase in all extracellular matrix (ECM) proteins was observed when the gonadal hormones were used concomitantly. Significant decrease in the amount of ECM proteins was observed on use of GnRH-a, LA and cetrorelix, with 24-hour exposure. Both the compounds also significantly decreased ECM protein concentration despite the presence of E2 or both gonadal hormones. This study demonstrates that GnRH-a directly affect the gonadal hormone-regulated collagen-1, fibronectin, and versican production in their presence. These findings suggest that localized therapy with GnRH-a may inhibit leiomyoma growth even in the presence of endogenous gonadal hormone exposure, thereby providing a mechanism to eliminate the hypoestrogenic side effects associated with GnRH-a therapy. Published by Elsevier Inc.

In vivo investigation on connective tissue healing to polished surfaces with different surface wettability.

PubMed

Kloss, Frank R; Steinmüller-Nethl, Doris; Stigler, Robert G; Ennemoser, Thomas; Rasse, Michael; Hächl, Oliver

2011-07-01

Connective tissue in contact to transgingival/-dermal implants presents itself as tight scar formation. Although rough surfaces support the attachment they increase bacterial colonisation as well. In contrast to surface roughness, little is known about the influence of surface wettability on soft-tissue healing in vivo. We therefore investigated the influence of different surface wettabilities on connective tissue healing at polished implant surfaces in vivo. Three polished experimental groups (titanium, titanium coated with hydrophobic nano-crystalline diamond (H-NCD) and titanium coated with hydrophilic nano-crystalline diamond (O-NCD) were inserted into the subcutaneous connective tissue of the abdominal wall of 24 rats. Animals were sacrificed after 1 and 4 weeks resulting in eight specimen per group per time point. Specimen were subjected to histological evaluation (van Giesson's staining) and immunohistochemistry staining for proliferating cell nuclear antigen (PCNA), fibronectin and tumour necrosis factor-alpha (TNF-α). Histological evaluation revealed dense scar formation at the titanium and H-NCD surfaces. In contrast, the connective tissue was loose at the O-NCD surface with a significantly higher number of cells after 4 weeks. O-NCD demonstrated a strong expression of PCNA and fibronectin but a weak expression of TNF-α. In contrast, the PCNA and fibronectin expression was low at the titanium and H-NCD, with a strong signal of TNF-α at the H-NCD surface. Hydrophilicity influences the connective tissue healing at polished implant surfaces in vivo positively. The attachment of connective tissue and the number of cells in contact to the surface were increased. Moreover, the inflammatory response is decreased at the hydrophilic surface. © 2010 John Wiley & Sons A/S.

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

PubMed

Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis.

PubMed

Larzabal, L; de Aberasturi, A L; Redrado, M; Rueda, P; Rodriguez, M J; Bodegas, M E; Montuenga, L M; Calvo, A

2014-02-04

TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels. We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.

Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption ▿

PubMed Central

Xu, Chun-Ping; Boks, Niels P.; de Vries, Joop; Kaper, Hans J.; Norde, Willem; Busscher, Henk J.; van der Mei, Henny C.

2008-01-01

Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow. PMID:18952882

TGFβ1-mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-02-28

Alternative splicing is a fundamental phenomenon to build protein diversity in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin protein present in the extra cellular matrix (ECM) in renal fibrosis. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. TGFβ1 is a strong stimulator of ECM proteins in renal injury. In this study, we have investigated alternative splicing of EDA+ Fn in human podocytes in response to TGFβ1. We have performed western blotting and immunofluorescence to characterise the expression of the EDA+Fn protein, real-time PCR for RNA expression and RT-PCR to look for alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We used TGFβ1 as a stimulator and SB431542 and SRPIN340 for inhibitory studies. In this work, for the first time we have demonstrated in human podocytes culture EDA+Fn is expressed in the basal condition and TGFβ1 2.5ng/ml induced the Fn mRNA and EDA+Fn protein expression demonstrated by real-time PCR, western blotting and immunofluorescence. TGFβ1 2.5ng/ml induced the alternative splicing of EDA+Fn shown by conventional RT-PCR. Studies with ALK5 inhibitor SB431542 and SRPIN340 show that TGFβ1 induced alternative splicing of EDA+Fn was by the ALK5 receptor and the SR proteins.  In human podocytes culture, alternative splicing of EDA+Fn occurs at basal conditions and TGFβ1 further induced the alternative splicing of EDA+Fn via ALK5 receptor activation and SR proteins. This is the first evidence of basal and TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture.

Origin-Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion.

PubMed

Sheriff, Lozan; Alanazi, Asma; Ward, Lewis S C; Ward, Carl; Munir, Hafsa; Rayes, Julie; Alassiri, Mohammed; Watson, Steve P; Newsome, Phil N; Rainger, G E; Kalia, Neena; Frampton, Jon; McGettrick, Helen M; Nash, Gerard B

2018-02-28

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018. © 2018 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

PubMed Central

Arregui, Carlos O.; Balsamo, Janne; Lilien, Jack

1998-01-01

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. PMID:9813103

Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model.

PubMed

Dawson, R A; Goberdhan, N J; Freedlander, E; MacNeil, S

1996-03-01

The aim of this study was to investigate whether prior culture of cells on ECM proteins might positively influence the performance of keratinocytes when cells are transferred to a dermal in vitro wound bed model. Keratinocytes were cultured using a method for producing cultured epithelial autografts for severely burned patients (essentially using Green's medium, a mitogen-rich medium containing fetal calf serum, cholera toxin, EGF, insulin, transferrin and triiodothyronine). Cells were cultured either on irradiated 3T3 fibroblasts (as in the standard Rheinwald and Green technique) or, alternatively, on collagen I, collagen IV, matrigel, RGD, vitronectin or fibronectin. Under these conditions matrigel, collagen I and IV enhanced initial attachment, RGD, vitronectin, fibronectin and irradiated 3T3 fibroblasts did not. Proliferation of cells was positively influenced by matrigel, collagen I and IV and irradiated 3T3 fibroblasts; of these, however, only matrigel and 3T3 fibroblasts had sustained significant effects on keratinocyte proliferation over 4 days. Cells on fibronectin showed significantly reduced proliferation. An acellular non-viable dermis was then used to mimic the homograft allodermis onto which cultured epithelial autograft sheets are grafted clinically and cells cultured on the various ECM proteins for 96 h were transferred to this in vitro wound model. None of the substrates enhanced keratinocyte performance on this model. It was concluded that under these conditions some ECM proteins can significantly affect keratinocyte attachment and, to a lesser extent, proliferation but that the culture of keratinocytes on these ECM proteins does not appear to confer any lasting benefit to the attachment of these keratinocytes to an in vitro wound-bed model.

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices.

PubMed

Sharma, Manjinder; Dubey, Pawan K; Kumar, Rajesh; Nath, Amar; Kumar, G Sai; Sharma, G Taru

2013-05-01

Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

Identification of a Novel Virulence Determinant with Serum Opacification Activity in Streptococcus suis

PubMed Central

Baums, Christoph G.; Kaim, Ute; Fulde, Marcus; Ramachandran, Girish; Goethe, Ralph; Valentin-Weigand, Peter

2006-01-01

Streptococcus suis serotype 2 is a porcine and human pathogen with adhesive and invasive properties. In other streptococci, large surface-associated proteins (>100 kDa) of the MSCRAMM family (microbial surface components recognizing adhesive matrix molecules) are key players in interactions with host tissue. In this study, we identified a novel opacity factor of S. suis (OFS) with structural homology to members of the MSCRAMM family. The N-terminal region of OFS is homologous to the respective regions of fibronectin-binding protein A (FnBA) of Streptococcus dysgalactiae and the serum opacity factor (SOF) of Streptococcus pyogenes. Similar to these two proteins, the N-terminal domain of OFS opacified horse serum. Serum opacification activity was detectable in sodium dodecyl sulfate extracts of wild-type S. suis but not in extracts of isogenic ofs knockout mutants. Heterologous expression of OFS in Lactococcus lactis demonstrated that a high level of expression of OFS is sufficient to provide surface-associated serum opacification activity. Furthermore, serum opacification could be inhibited by an antiserum against recombinant OFS. The C-terminal repetitive sequence elements of OFS differed significantly from the respective repeat regions of FnBA and SOF as well as from the consensus sequence of the fibronectin-binding repeats of MSCRAMMs. Accordingly, fibronectin binding was not detectable in recombinant OFS. To investigate the putative function of OFS in the pathogenesis of invasive S. suis diseases, piglets were experimentally infected with an isogenic mutant strain in which the ofs gene had been knocked out by an in-frame deletion. The mutant was severely attenuated in virulence but not in colonization, demonstrating that OFS represents a novel virulence determinant of S. suis. PMID:17057090

Mesenchymal stem cells protect against obstruction-induced renal fibrosis by decreasing STAT3 activation and STAT3-dependent MMP-9 production.

PubMed

Matsui, Futoshi; Babitz, Stephen A; Rhee, Audrey; Hile, Karen L; Zhang, Hongji; Meldrum, Kirstan K

2017-01-01

STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mesenchymal stem cell (MSC)-induced renoprotection during renal fibrosis remains unknown. We hypothesized that MSCs protect against obstruction-induced renal fibrosis by downregulating STAT3 activation and STAT3-induced matrix metalloproteinase-9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 × 10 6 /rat) immediately before sham operation or induction of unilateral ureteral obstruction (UUO). The kidneys were harvested after 4 wk and analyzed for collagen I and III gene expression, collagen deposition (Masson's trichrome), fibronectin, α-smooth muscle actin, active STAT3 (p-STAT3), MMP-9, and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) expression. In a separate arm, the STAT3 inhibitor S3I-201 (10 mg/kg) vs. vehicle was administered to rats intraperitoneally just after induction of UUO and daily for 14 days thereafter. The kidneys were harvested after 2 wk and analyzed for p-STAT3 and MMP-9 expression, and collagen and fibronectin deposition. Renal obstruction induced a significant increase in collagen, fibronectin, α-SMA, p-STAT3, MMP-9, and TIMP-1 expression while exogenously administered MSCs significantly reduced these indicators of obstruction-induced renal fibrosis. STAT3 inhibition with S3I-201 significantly reduced obstruction-induced MMP-9 expression and tubulointerstitial fibrosis. These results demonstrate that MSCs protect against obstruction-induced renal fibrosis, in part, by decreasing STAT3 activation and STAT3-dependent MMP-9 production. Copyright © 2017 the American Physiological Society.

Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells

PubMed Central

Chaturvedi, Lakshmi S.; Marsh, Harold M.

2011-01-01

Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mDia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mDia1 by smart-pool double-stranded short-interfering RNAs (siRNA) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mDia1 siRNAs. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mDia1, and FAK reduction by siRNA blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mDia1 reduction by siRNA also blocked strain-induced FAK-Tyr925, p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mDia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation. PMID:21849669

CCAAT/Enhancer Binding Protein–α Regulates the Protease/Antiprotease Balance Required for Bronchiolar Epithelium Regeneration

PubMed Central

Sato, Atsuyasu; Xu, Yan; Whitsett, Jeffrey A.

2012-01-01

Many transcription factors that regulate lung morphogenesis during development are reactivated to mediate repairs of the injured adult lung. We hypothesized that CCAAT/enhancer binding protein–α (C/EBPα), a transcription factor critical for perinatal lung maturation, regulates genes required for the normal repair of the bronchiolar epithelium after injury. Transgenic CebpαΔ/Δ mice, in which Cebpa was conditionally deleted from Clara cells and Type II cells after birth, were used in this study. Airway injury was induced in mice by the intraperitoneal administration of naphthalene to ablate bronchiolar epithelial cells. Although the deletion of C/EBPα did not influence lung structure and function under unstressed conditions, C/EBPα was required for the normal repair of terminal bronchiolar epithelium after naphthalene injury. To identify cellular processes that are influenced by C/EBPα during repair, mRNA microarray was performed on terminal bronchiolar epithelial cells isolated by laser-capture microdissection. Normal repair of the terminal bronchiolar epithelium was highly associated with the mRNAs regulating antiprotease activities, and their induction required C/EBPα. The defective deposition of fibronectin in CebpαΔ/Δ mice was associated with increased protease activity and delayed differentiation of FoxJ1-expressing ciliated cells. The fibronectin and ciliated cells were restored by the intratracheal treatment of CebpαΔ/Δ mice with the serine protease inhibitor. In conclusion, C/EBPα regulates the expression of serine protease inhibitors that are required for the normal increase of fibronectin and the restoration of ciliated cells after injury. Treatment with serine protease inhibitor may aid in the recovery of injured bronchiolar epithelial cells, and prevent common chronic lung diseases. PMID:22652201

Dietary docosahexaenoic acid ameliorates, but rapeseed oil and safflower oil accelerate renal injury in stroke-prone spontaneously hypertensive rats as compared with soybean oil, which is associated with expression for renal transforming growth factor-beta, fibronectin and renin.

PubMed

Miyazaki, M; Takemura, N; Watanabe, S; Hata, N; Misawa, Y; Okuyama, H

2000-01-03

We have noted that n-3 fatty acid-rich oils, such as fish oil, perilla oil and flaxseed oil as well as ethyl docosahexaenoate (DHA) prolonged the survival time of stroke-prone spontaneously hypertensive rats (SHRSP) rats by approximately 10% as compared with linoleate (n-6)-rich safflower oil. Rapeseed oil with a relatively low n-6/n-3 ratio unusually shortened the survival time by approximately 40%, suggesting the presence of minor components unfavorable to SHRSP rats. This study examined the effects of dietary oils and DHA on renal injury and gene expression related to renal injury in SHRSP rats. Rats fed rapeseed oil- and safflower oil-supplemented diets developed more severe proteinuria than those fed soybean oil-supplemented diet used as a control, but there were no significant differences in blood pressure. In contrast, the DHA-supplemented diet inhibited the development of proteinuria and suppressed hypertension. The mRNA levels for renal TGF-beta, fibronectin and renin were higher in the rapeseed oil and safflower oil groups after 9 weeks of feeding of the experimental diet than in the soybean oil and DHA groups. The fatty acid composition of kidney phospholipids was markedly affected by these diets. These results indicate that the renal injury observed in the groups fed safflower oil with a high n-6/n-3 ratio and rapeseed oil with presumed minor components is accompanied by increased expression of the TGF-beta, renin and fibronectin genes, and that dietary DHA suppresses renal injury and gene expression as compared with soybean oil.

Characterization of a highly conserved human homolog to the chicken neural cell surface protein Bravo/Nr-CAM that maps to chromosome band 7q31

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lane, R.P.; Vielmetter, J.; Dreyer, W.J.

1996-08-01

The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Igmore » domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescence in situ hybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1-q31.2. This chromosomal locus has been previously identified as containing a tumore suppressor candidate gene commonly deleted in certain human cancer tissues. 38 refs., 5 figs.« less

Outcomes and predictive tests from a dedicated specialist clinic for women at high risk of preterm labour: A ten year audit.

PubMed

Hughes, Kelly; Sim, Shirlene; Roman, Alina; Michalak, Kasia; Kane, Stefan; Sheehan, Penelope

2017-08-01

Preterm birth continues to be a major cause of infant morbidity and mortality worldwide, but advances have recently been made in its prediction and prevention. A short cervix (<25 mm) in the second trimester on transvaginal ultrasound scan and fetal fibronectin are important predictive tests. For over ten years, the Preterm Labour Clinic at the Royal Women's Hospital, Melbourne, Australia has provided care for women at high risk of preterm birth, including those with a previous preterm birth, previous cervical surgery, uterine malformation or incidental finding of short cervix at routine ultrasound. The purpose of this study was to review this clinic's outcomes for the first decade. This was a retrospective cohort study of all referrals to the Preterm Labour Clinic during the period 2004-2013 inclusive. Seven hundred and fifty-six cases met the study inclusion criteria of appropriate risk factor, singleton pregnancy, surveillance undertaken and outcome data available. The preterm birth rate (<37 weeks) was 21.4%. The rate of preterm birth by year decreased significantly when adjusted for risk (P = 0.002). A short cervix was diagnosed in 32% of the sample, and positively correlated with lower gestational age at delivery. Fetal fibronectin and serum alkaline phosphatase were independent predictors of preterm birth <34 weeks and <37 weeks. The adjusted preterm birth rate at the Royal Women's Hospital's Preterm Labour Clinic has decreased significantly over the decade studied. Positive fetal fibronectin at 26 weeks and elevated serum alkaline phosphatase are independent predictors of preterm birth. © 2017 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium.

PubMed

Shynlova, Oksana; Mitchell, Jennifer A; Tsampalieros, Anne; Langille, B Lowell; Lye, Stephen J

2004-04-01

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.

A Multi-Scale Study on the Role of Trace Metals on Physiological and Pathological Mineralization

NASA Astrophysics Data System (ADS)

Rammelkamp, Derek

The work in this thesis provides mulit-scale contributions towards understanding the effects of trace metals on the pathological mineralization process relating to both the development of healthy bone tissue, the diseased state of osteoporosis, and microcalcifications which develop in breast cancers. A protein level study was performed on ECM protein fibronectin, which plays a role in cell adhesion. The protein studies showed zinc interactions with fibronectin and its fragment, anastellin, to influence protein structure. Zinc is also shown to decrease cell migration in vitro, which may be influenced by changes in fibronectin ECM structure. The effects of osteoporosis on micronutrient composition in vivo were examined using the technique of x-ray fluorescence (XRF) in an ovariectomized rat model. Compared to healthy bone, subtle difference are observed in zinc and iron in osteoporotic rat bones, showing micronutrients may play an important role in healthy bone regulation. Effects of micronutrient zinc was used to inhibit microcalcification formation in breast cancers. Microcalcifications have been linked malignancy of breast cancers, but the process of microcalcification formation has yet to be well understood. In this work, exogenous zinc is used to inhibit microcalcification formation, and metastatic potential in both a 2D and 3D spheroid environment. A novel in vitro self-assembled three dimensional multi-cellular tumor spheroid (MCTS) model for the study of breast cancer microcalcifications was developed for this experiment. A MCTS model for studying breast cancer microcalcifications has potential to be used in drug discovery, or for basic research applications studying mechanisms of microcalcification formation, which are still not fully understood. Taken together this study uses a multi-scale approach to gain a better understanding of micronutrients involved in pathological mineralization.

Hsp90 Binds Directly to Fibronectin (FN) and Inhibition Reduces the Extracellular Fibronectin Matrix in Breast Cancer Cells

PubMed Central

Kenyon, Amy; Dhanani, Karim C. H.; Prinsloo, Earl; Edkins, Adrienne L.

2014-01-01

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis. PMID:24466266

Hsp90 binds directly to fibronectin (FN) and inhibition reduces the extracellular fibronectin matrix in breast cancer cells.

PubMed

Hunter, Morgan C; O'Hagan, Kyle L; Kenyon, Amy; Dhanani, Karim C H; Prinsloo, Earl; Edkins, Adrienne L

2014-01-01

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.

Disruption of β-catenin/CBP signaling inhibits human airway epithelial-mesenchymal transition and repair.

PubMed

Moheimani, Fatemeh; Roth, Hollis M; Cross, Jennifer; Reid, Andrew T; Shaheen, Furquan; Warner, Stephanie M; Hirota, Jeremy A; Kicic, Anthony; Hallstrand, Teal S; Kahn, Michael; Stick, Stephen M; Hansbro, Philip M; Hackett, Tillie-Louise; Knight, Darryl A

2015-11-01

The epithelium of asthmatics is characterized by reduced expression of E-cadherin and increased expression of the basal cell markers ck-5 and p63 that is indicative of a relatively undifferentiated repairing epithelium. This phenotype correlates with increased proliferation, compromised wound healing and an enhanced capacity to undergo epithelial-mesenchymal transition (EMT). The transcription factor β-catenin plays a vital role in epithelial cell differentiation and regeneration, depending on the co-factor recruited. Transcriptional programs driven by the β-catenin/CBP axis are critical for maintaining an undifferentiated and proliferative state, whereas the β-catenin/p300 axis is associated with cell differentiation. We hypothesized that disrupting the β-catenin/CBP signaling axis would promote epithelial differentiation and inhibit EMT. We treated monolayer cultures of human airway epithelial cells with TGFβ1 in the presence or absence of the selective small molecule ICG-001 to inhibit β-catenin/CBP signaling. We used western blots to assess expression of an EMT signature, CBP, p300, β-catenin, fibronectin and ITGβ1 and scratch wound assays to assess epithelial cell migration. Snai-1 and -2 expressions were determined using q-PCR. Exposure to TGFβ1 induced EMT, characterized by reduced E-cadherin expression with increased expression of α-smooth muscle actin and EDA-fibronectin. Either co-treatment or therapeutic administration of ICG-001 completely inhibited TGFβ1-induced EMT. ICG-001 also reduced the expression of ck-5 and -19 independent of TGFβ1. Exposure to ICG-001 significantly inhibited epithelial cell proliferation and migration, coincident with a down regulation of ITGβ1 and fibronectin expression. These data support our hypothesis that modulating the β-catenin/CBP signaling axis plays a key role in epithelial plasticity and function. Copyright © 2015 Elsevier Ltd. All rights reserved.

Serpina3n accelerates tissue repair in a diabetic mouse model of delayed wound healing.

PubMed

Hsu, I; Parkinson, L G; Shen, Y; Toro, A; Brown, T; Zhao, H; Bleackley, R C; Granville, D J

2014-10-09

Chronic, non-healing wounds are a major complication of diabetes and are characterized by chronic inflammation and excessive protease activity. Although once thought to function primarily as a pro-apoptotic serine protease, granzyme B (GzmB) can also accumulate in the extracellular matrix (ECM) during chronic inflammation and cleave ECM proteins that are essential for proper wound healing, including fibronectin. We hypothesized that GzmB contributes to the pathogenesis of impaired diabetic wound healing through excessive ECM degradation. In the present study, the murine serine protease inhibitor, serpina3n (SA3N), was administered to excisional wounds created on the dorsum of genetically induced type-II diabetic mice. Wound closure was monitored and skin wound samples were collected for analyses. Wound closure, including both re-epithelialization and contraction, were significantly increased in SA3N-treated wounds. Histological and immunohistochemical analyses of SA3N-treated wounds revealed a more mature, proliferative granulation tissue phenotype as indicated by increased cell proliferation, vascularization, fibroblast maturation and differentiation, and collagen deposition. Skin homogenates from SA3N-treated wounds also exhibited greater levels of full-length intact fibronectin compared with that of vehicle wounds. In addition, GzmB-induced detachment of mouse embryonic fibroblasts correlated with a rounded and clustered phenotype that was prevented by SA3N. In summary, topical administration of SA3N accelerated wound healing. Our findings suggest that GzmB contributes to the pathogenesis of diabetic wound healing through the proteolytic cleavage of fibronectin that is essential for normal wound closure, and that SA3N promotes granulation tissue maturation and collagen deposition.

Osteograft, plastic material for regenerative medicine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zaidman, A. M., E-mail: AZaydman@niito.ru; Korel, A. V., E-mail: AKorel@niito.ru; Shchelkunova, E. I., E-mail: EShelkunova@niito.ru

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14–30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissuemore » specificity of the developed osteograft.« less

Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix.

PubMed

Kubow, Kristopher E; Vukmirovic, Radmila; Zhe, Lin; Klotzsch, Enrico; Smith, Michael L; Gourdon, Delphine; Luna, Sheila; Vogel, Viola

2015-08-14

Despite the crucial role of extracellular matrix (ECM) in directing cell fate in healthy and diseased tissues--particularly in development, wound healing, tissue regeneration and cancer--the mechanisms that direct the assembly and regulate hierarchical architectures of ECM are poorly understood. Collagen I matrix assembly in vivo requires active fibronectin (Fn) fibrillogenesis by cells. Here we exploit Fn-FRET probes as mechanical strain sensors and demonstrate that collagen I fibres preferentially co-localize with more-relaxed Fn fibrils in the ECM of fibroblasts in cell culture. Fibre stretch-assay studies reveal that collagen I's Fn-binding domain is responsible for the mechano-regulated interaction. Furthermore, we show that Fn-collagen interactions are reciprocal: relaxed Fn fibrils act as multivalent templates for collagen assembly, but once assembled, collagen fibres shield Fn fibres from being stretched by cellular traction forces. Thus, in addition to the well-recognized, force-regulated, cell-matrix interactions, forces also tune the interactions between different structural ECM components.

Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor?

PubMed

Erickson, Harold P

2013-10-01

FNDC5 (fibronectin domain-containing [protein] 5) was initially discovered and characterized by two groups in 2002. In 2011 FNDC5 burst into prominence as the parent of irisin, a small protein containing the fibronectin type III domain. Irisin was proposed to be secreted by skeletal muscle cells in response to exercise, and to circulate to fat tissue where it induced a transition to brown fat. Since brown fat results in dissipation of energy, this pathway is of considerable interest for metabolism and obesity. Here I review the original discoveries of FNDC5 and the more recent discovery of irisin. I note in particular three problems in the characterization of irisin: the antibodies used to detect irisin in plasma lack validity; the recombinant protein used to demonstrate activity in cell culture was severely truncated; and the degree of shedding of soluble irisin from the cell surface has not been quantitated. The original discovery proposing that FNDC5 may be a transmembrane receptor may deserve a new look.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

PubMed Central

Yu, Jiachao; Zhu, Zihua; Yu, Xiao-Ying

2016-01-01

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time. PMID:26966995

Evolution of sfbI Encoding Streptococcal Fibronectin-Binding Protein I: Horizontal Genetic Transfer and Gene Mosaic Structure

PubMed Central

Towers, Rebecca J.; Fagan, Peter K.; Talay, Susanne R.; Currie, Bart J.; Sriprakash, Kadaba S.; Walker, Mark J.; Chhatwal, Gursharan S.

2003-01-01

Streptococcal fibronectin-binding protein is an important virulence factor involved in colonization and invasion of epithelial cells and tissues by Streptococcus pyogenes. In order to investigate the mechanisms involved in the evolution of sfbI, the sfbI genes from 54 strains were sequenced. Thirty-four distinct alleles were identified. Three principal mechanisms appear to have been involved in the evolution of sfbI. The amino-terminal aromatic amino acid-rich domain is the most variable region and is apparently generated by intergenic recombination of horizontally acquired DNA cassettes, resulting in a genetic mosaic in this region. Two distinct and divergent sequence types that shared only 61 to 70% identity were identified in the central proline-rich region, while variation at the 3′ end of the gene is due to deletion or duplication of defined repeat units. Potential antigenic and functional variabilities in SfbI imply significant selective pressure in vivo with direct implications for the microbial pathogenesis of S. pyogenes. PMID:14662917

Fibronectin regulates Wnt7a signaling and satellite cell expansion

PubMed Central

Bentzinger, C. Florian; Wang, Yu Xin; von Maltzahn, Julia; Soleimani, Vahab D.; Yin, Hang; Rudnicki, Michael A.

2012-01-01

SUMMARY The influence of the extracellular matrix (ECM) within the stem cell niche remains poorly understood. We found that Syndecan-4 (Sdc4) and Frizzled-7 (Fzd7) form a co-receptor complex in satellite cells and that binding of the ECM glycoprotein Fibronectin (FN) to Sdc4 stimulates the ability of Wnt7a to induce the symmetric expansion of satellite stem cells. Newly activated satellite cells dynamically remodel their niche by transient high-level expression of FN. Knockdown of FN in prospectively isolated satellite cells severely impaired their ability to repopulate the satellite cell niche. Conversely, in vivo over-expression of FN with Wnt7a dramatically stimulated the expansion of satellite stem cells in regenerating muscle. Therefore, activating satellite cells remodel their niche through autologous expression of FN that provides feedback to stimulate Wnt7a signaling through the Fzd7/Sdc4 co-receptor complex. Thus, FN and Wnt7a together regulate the homeostatic levels of satellite stem cells and satellite myogenic cells during regenerative myogenesis. PMID:23290138

Effect of clindamycin treatment on vaginal inflammatory markers in pregnant women with bacterial vaginosis and a positive fetal fibronectin test.

PubMed

Diaz-Cueto, Laura; Dominguez-Lopez, Pablo; Tena-Alavez, Gilberto; Cuica-Flores, Adrian; Rosales-Ortiz, Sergio; Arechavaleta-Velasco, Fabian

2009-11-01

To compare the levels of interleukin (IL)-1beta, IL-6, and matrix metalloproteinase (MMP)-8 in the vaginal secretions of pregnant women with a positive fetal fibronectin (fFN) test result with or without asymptomatic bacterial vaginosis (BV) before and after treatment with oral clindamycin. A prospective cohort study was conducted among 43 pregnant women with a positive fFN test result. All patients were treated with clindamycin, and the pre- and post-treatment levels of IL-1beta, IL-6, and MMP-8 were compared. Before treatment, levels of IL-1beta and MMP-8 were significantly higher in women with BV compared with women without BV (P<0.05). Vaginal levels of IL-1beta and IL-6, but not MMP-8, decreased after treatment in pregnant women with BV. The inability of clindamycin to decrease MMP-8 vaginal levels may explain why it is ineffective in reducing preterm birth in pregnant women with positive fFN and BV.

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin.

PubMed

Erdogan, Begum; Ao, Mingfang; White, Lauren M; Means, Anna L; Brewer, Bryson M; Yang, Lijie; Washington, M Kay; Shi, Chanjuan; Franco, Omar E; Weaver, Alissa M; Hayward, Simon W; Li, Deyu; Webb, Donna J

2017-11-06

Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF-cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α-mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration. © 2017 Erdogan et al.

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin

PubMed Central

Ao, Mingfang; White, Lauren M.; Means, Anna L.; Yang, Lijie; Washington, M. Kay; Franco, Omar E.; Li, Deyu; Webb, Donna J.

2017-01-01

Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF–cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α–mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration. PMID:29021221

Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling.

PubMed

Hoffmann, Celine; Leroy-Dudal, Johanne; Patel, Salima; Gallet, Olivier; Pauthe, Emmanuel

2008-01-01

Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.

Detachment strength of human osteoblasts cultured on hydroxyapatite with various surface roughness. Contribution of integrin subunits.

PubMed

Kokkinos, Petros A; Koutsoukos, Petros G; Deligianni, Despina D

2012-06-01

Hydroxyapatite (HA) has been widely used as a bone substitute in dental, maxillofacial and orthopaedic surgery and as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. The aim of the present study was to investigate the osteoblastic adhesion strength on HA substrata with different surface topography and biochemistry (pre-adsorption of fibronectin) after blocking of specific integrin subunits with monoclonal antibodies. Stoichiometric HA was prepared by precipitation followed by ageing and characterized by SEM, EDX, powder XRD, Raman spectroscopy, TGA, and specific surface area analysis. Human bone marrow derived osteoblasts were cultured on HA disc-shaped substrata which were sintered and polished resulting in two surface roughness grades. For attachment evaluation, cells were incubated with monoclonal antibodies and seeded for 2 h on the substrata. Cell detachment strength was determined using a rotating disc device. Cell detachment strength was surface roughness, fibronectin preadsorption and intergin subunit sensitive.

Biocompatibility of helicoidal multilamellar arginine-glycine-aspartic acid-functionalized silk biomaterials in a rabbit corneal model.

PubMed

Wang, Liqiang; Ma, Ruijue; Du, Gaiping; Guo, Huiling; Huang, Yifei

2015-01-01

Silk proteins represent a unique choice in the selection of biomaterials that can be used for corneal tissue engineering and regenerative medical applications. We implanted helicoidal multilamellar arginine-glycine-aspartic acid-functionalized silk biomaterials into the corneal stroma of rabbits, and evaluated its biocompatibility. The corneal tissue was examined after routine hematoxylin-eosin staining, immunofluorescence for collagen I and III, and fibronectin, and scanning electron microscopy. The silk films maintained their integrity and transparency over the 180-day experimental period without causing immunogenic and neovascular responses or degradation of the rabbit corneal stroma. Collagen I increased, whereas Collagen III and fibronectin initially increased and then gradually decreased. The extracellular matrix deposited on the surface of the silk films, tightly adhered to the biomaterial. We have shown this kind of silk film graft has suitable biocompatibility with the corneal stroma and is an initial step for clinical trials to evaluate this material as a transplant biomaterial for keratoplasty tissue constructs. © 2014 Wiley Periodicals, Inc.

[Effects of fibronectin on cytodifferentiation (T56) of human squamous cell carcinoma of tongue].

PubMed

Huang, Y; Yang, F

1994-12-01

Fibronectin (FN) are large glycoproteins that have been implicated in a wide variety of cellular properties, including cell adhesion, morphology, cytoskeletal organization, migration, differentiation, and oncogenic transformation. T56 cells were treated with 20, 50, and 100 micrograms/ml of FN for 48 h, and the changes in cells were analysed qualitatively and quantitatively with the methods of transmission electron microscopy, enzyme cytochemistry, and cell electrophoresis, etc. The studies were made to test the effects of FN on T56 cells in biological behaviour in terms of cell growth, multiplication, cell metabolism, cell electrophoresis and surface morphology. The results indicated that FN could partially restore T56 cell normal epidermic cell's phenotype, inhibit cell mitosis, increase contact of cells, decrease the number of microvilli and ruffles, and promote cell oxybiotic metabolism. These results suggest that FN might relate in many aspects to the biological behaviour of T56 cell and could affect changes in the behaviour.

Fibronectin domains of extracellular matrix molecule tenascin-C modulate hippocampal learning and synaptic plasticity.

PubMed

Strekalova, Tatyana; Sun, Mu; Sibbe, Mirjam; Evers, Matthias; Dityatev, Alexander; Gass, Peter; Schachner, Melitta

2002-09-01

The extracellular matrix molecule tenascin-C (TN-C) has been shown to be involved in hippocampal synaptic plasticity in vitro. Here, we describe a deficit in hippocampus-dependent contextual memory in TN-C-deficient mice using the step-down avoidance paradigm. We further show that a fragment of TN-C containing the fibronectin type-III repeats 6-8 (FN6-8), but not a fragment containing repeats 3-5, bound to pyramidal and granule cell somata in the hippocampal formation of C57BL/6J mice and repelled axons of pyramidal neurons when presented as a border in vitro. Injection of the FN6-8 fragment into the hippocampus inhibited retention of memory in the step-down paradigm and reduced levels of long-term potentiation in the CA1 region of the hippocampus. In summary, our data show that TN-C is involved in hippocampus-dependent contextual memory and synaptic plasticity and identify the FN6-8 domain as one of molecular determinants mediating these functions.

An iridium oxide microelectrode for monitoring acute local pH changes of endothelial cells.

PubMed

Ng, Shu Rui; O'Hare, Danny

2015-06-21

pH sensors were fabricated by anodically electrodepositing iridium oxide films (AEIROFs) onto microelectrodes on chips and coated with poly(ethyleneimine) (PEI) for mechanical stability. These demonstrate super-Nernstian response to pH from pH 4.0 to 7.7 in chloride-free phosphate buffer. The surface of the chip was coated with fibronectin for the attachment of porcine aortic endothelial cells (PAECs). The working capability of the pH sensor for monitoring acute local pH changes was investigated by stimulating the PAECs with thrombin. Our results show that thrombin induced acute extracellular acidification of PAECs and dissolution of fibronectin, causing the local pH to decrease. The use of PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, reduced extracellular acidification and an increase in local pH was observed. This study shows that our pH sensors can facilitate the investigation of acute cellular responses to stimulation by monitoring the real-time, local pH changes of cells attached to the sensors.

Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy.

PubMed

Chen, Cheng; Gong, Wenyan; Li, Changzheng; Xiong, Fengxiao; Wang, Shaogui; Huang, Junying; Wang, Yu; Chen, Zhiquan; Chen, Qiuhong; Liu, Peiqing; Lan, Tian; Huang, Heqing

2017-10-03

Activation of sphingosine kinase 1 (SphK1) signaling pathway mediates fibronectin (FN) upregulation in glomerular mesangial cells (GMCs) under high glucose (HG) condition. However, the roles of SphK1 in advanced glycation end products (AGEs)-induced DN have not been elucidated. Here we show that AGEs upregulated FN and SphK1 and SphK1 activity. Inhibition of SphK1 signaling attenuated AGEs-induced FN synthesis in GMCs. Inhibition of AGE receptor (RAGE) signaling reduced the upregulation of FN and SphK1 and SphK1 activity in GMCs induced by AGEs. Treatment of aminoguanidine ameliorates the renal injury and fibrosis in STZ-induced diabetic mice and attenuated SphK1 expression and activity in diabetic mouse kidneys. The renal injury and fibrosis in diabetic SphK1 -/- mice was significantly attenuated than WT mice. Furthermore, AGEs upregulated SphK1 by reducing its degradation and prolonging its half-life. SphK1 mediates AGEs-induced FN synthesis in GMCs and diabetic mice under hyperglycemic condition .

ATX-LPA1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation

PubMed Central

Nishioka, Tatsuji; Arima, Naoaki; Kano, Kuniyuki; Hama, Kotaro; Itai, Eriko; Yukiura, Hiroshi; Kise, Ryoji; Inoue, Asuka; Kim, Seok-Hyung; Solnica-Krezel, Lilianna; Moolenaar, Wouter H.; Chun, Jerold; Aoki, Junken

2016-01-01

The lipid mediator lysophosphatidic acid (LPA) signals via six distinct G protein-coupled receptors to mediate both unique and overlapping biological effects, including cell migration, proliferation and survival. LPA is produced extracellularly by autotaxin (ATX), a secreted lysophospholipase D, from lysophosphatidylcholine. ATX-LPA receptor signaling is essential for normal development and implicated in various (patho)physiological processes, but underlying mechanisms remain incompletely understood. Through gene targeting approaches in zebrafish and mice, we show here that loss of ATX-LPA1 signaling leads to disorganization of chondrocytes, causing severe defects in cartilage formation. Mechanistically, ATX-LPA1 signaling acts by promoting S-phase entry and cell proliferation of chondrocytes both in vitro and in vivo, at least in part through β1-integrin translocation leading to fibronectin assembly and further extracellular matrix deposition; this in turn promotes chondrocyte-matrix adhesion and cell proliferation. Thus, the ATX-LPA1 axis is a key regulator of cartilage formation. PMID:27005960

Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa.

PubMed

Arduino, Paolo G; Carrozzo, Marco; Pagano, Marco; Broccoletti, Roberto; Scully, Crispian; Gandolfo, Sergio

2010-06-01

Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the pattern of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone.

Inhalation of Carbon Black Nanoparticles Aggravates Pulmonary Inflammation in Mice

PubMed Central

Saputra, Devina; Yoon, Jin-ha; Park, Hyunju; Heo, Yongju; Yang, Hyoseon; Lee, Eun Ji; Lee, Sangjin; Song, Chang-Woo; Lee, Kyuhong

2014-01-01

An increasing number of recent studies have focused on the impact of particulate matter on human health. As a model for atmospheric particulate inhalation, we investigated the effects of inhaled carbon black nanoparticles (CBNP) on mice with bleomycin-induced pulmonary fibrosis. The CNBPs were generated by a novel aerosolization process, and the mice were exposed to the aerosol for 4 hours. We found that CBNP inhalation exacerbated lung inflammation, as evidenced by histopathology analysis and by the expression levels of interleukin-6 protein, fibronectin, and interferon-γ mRNAs in lung tissues. Notably, fibronectin mRNA expression showed a statistically significant increase in expression after CBNP exposure. These data suggest that the concentration of CBNPs delivered (calculated to be 12.5 μg/m3) can aggravate lung inflammation in mice. Our results also suggest that the inhalation of ultrafine particles like PM 2.5 is an impactful environmental risk factor for humans, particularly in susceptible populations with predisposing lung conditions. PMID:25071917

Mena binds α5 integrin directly and modulates α5β1 function.

PubMed

Gupton, Stephanie L; Riquelme, Daisy; Hughes-Alford, Shannon K; Tadros, Jenny; Rudina, Shireen S; Hynes, Richard O; Lauffenburger, Douglas; Gertler, Frank B

2012-08-20

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell-cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue "LERER" repeats. In fibroblasts, the Mena-α5 complex was required for "outside-in" α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins.

Mena binds α5 integrin directly and modulates α5β1 function

PubMed Central

Riquelme, Daisy; Hughes-Alford, Shannon K.; Tadros, Jenny; Rudina, Shireen S.; O.Hynes, Richard; Lauffenburger, Douglas

2012-01-01

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell–cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue “LERER” repeats. In fibroblasts, the Mena–α5 complex was required for “outside-in” α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins. PMID:22908313

[Morphological features of the myometrium in connective tissue dysplasia in women with uterine inertia].

PubMed

Konovalov, P V; Mitrofanova, L B; Gorshkov, A N; Ovsyannikov, F A

2015-01-01

to reveal the morphological features of the lower uterine segment myometrium in connective tissue dysplasia (CTD) in women with uterine inertia. Histological, immunohistochemical (with antibodies against collagen types I and III, matrix metalloproteinases 1 and 9 (MMR-1, MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), fibronectin; fibulin-5, connexin-43), electron microscopic, and electron immunocytochemical studies with morphometry of myometrial fragments from 15 parturient women with CTD and uterine inertia (a study group) and those from 10 women without CTD (a control group). The myometrium in CTD exhibited the decreased expression of connextin-43, fibulin-5, TIMP-1, collagens types I and III with collagen type III predominance and the unchanged levels of fibronectin and MMP-1 and MMP-9. Electron microscopy and immunocytochemistry showed fewer intercellular contacts and the dramatically lower expression of connexin-43 than in the control. A set of found myometrial changes in women with uterine inertia is a manifestation of CTD.

Muscle development is disrupted in zebrafish embryos deficient for Fibronectin

PubMed Central

Snow, Chelsi J.; Peterson, Matthew T.; Khalil, Andre; Henry, Clarissa A.

2008-01-01

After somitogenesis, skeletal muscle precursors elongate into muscle fibers that anchor to the somite boundary, which becomes the myotome boundary. Fibronectin (Fn) is a major component of the extracellular matrix in both boundaries. Although Fn is required for somitogenesis, effects of Fn disruption on subsequent muscle development are unknown. We show fn knockdown disrupts myogenesis. Muscle morphogenesis is more disrupted in fn morphants than in a mutant where initial somite boundaries did not form, aei/deltaD. We quantified this disruption using the 2D Wavelet-Transform Modulus Maxima method, which uses the variation of intensity in an image with respect to the direction considered to characterize the structure in a cell lattice. We show that fibers in fn morphants are less organized than in aei/deltaD mutant embryos. Fast- and slow-twitch muscle lengths are also more frequently uncoupled. These data suggest fn may function to regulate fiber organization and limit fast-twitch muscle fiber length. PMID:18729220

Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

PubMed

Strachan, Lauren R; Condic, Maureen L

2004-11-08

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

Beneficial modulation from a high-purity caviar-derived homogenate on chronological skin aging.

PubMed

Marotta, Francesco; Polimeni, Ascanio; Solimene, Umberto; Lorenzetti, Aldo; Minelli, Emilio; Jain, Shalini; Rastmanesh, Reza; Sedriep, Sonia; Soresi, Vincenzo

2012-04-01

This study tested the activity of LD-1227, which contains a caviar-derived homogenate added with coenzyme Q(10) (CoQ(10))-selenium component (CaviarLieri(®), Lab-Dom, Switzerland), in aged human skin and its potential role on skin mitochondria function. Human dermal fibroblasts were obtained from healthy donors over 70 years old and treated with LD-1227 for 72 hr. As compared to baseline, LD-1227 caused a robust (>67%) collagen type I synthesis (p<0.001) and decreased fibronectin synthesis (p<0.05) with significant fibronectin messenger RNA (mRNA) downregulation (p<0.05, r=0.78). A significant collagen mRNA overexpression occurred with LD-1227 treatment (p<0.05). Mitochondria cytosolic adenosine triphosphate (ATP) level decreased in aged skin samples (p<0.05 vs. young control), but this phenomenon was reversed by LD-1227 (p<0.01). These data show that LD-1227 may modify the extracellular matrix milieu in aged skin and also beneficially affect mitochondrial function.

ATX-LPA1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation.

PubMed

Nishioka, Tatsuji; Arima, Naoaki; Kano, Kuniyuki; Hama, Kotaro; Itai, Eriko; Yukiura, Hiroshi; Kise, Ryoji; Inoue, Asuka; Kim, Seok-Hyung; Solnica-Krezel, Lilianna; Moolenaar, Wouter H; Chun, Jerold; Aoki, Junken

2016-03-23

The lipid mediator lysophosphatidic acid (LPA) signals via six distinct G protein-coupled receptors to mediate both unique and overlapping biological effects, including cell migration, proliferation and survival. LPA is produced extracellularly by autotaxin (ATX), a secreted lysophospholipase D, from lysophosphatidylcholine. ATX-LPA receptor signaling is essential for normal development and implicated in various (patho)physiological processes, but underlying mechanisms remain incompletely understood. Through gene targeting approaches in zebrafish and mice, we show here that loss of ATX-LPA1 signaling leads to disorganization of chondrocytes, causing severe defects in cartilage formation. Mechanistically, ATX-LPA1 signaling acts by promoting S-phase entry and cell proliferation of chondrocytes both in vitro and in vivo, at least in part through β1-integrin translocation leading to fibronectin assembly and further extracellular matrix deposition; this in turn promotes chondrocyte-matrix adhesion and cell proliferation. Thus, the ATX-LPA1 axis is a key regulator of cartilage formation.

Screening of gestational diabetes mellitus in early pregnancy by oral glucose tolerance test and glycosylated fibronectin: study protocol for an international, prospective, multicentre cohort trial.

PubMed

Huhn, E A; Fischer, T; Göbl, C S; Todesco Bernasconi, M; Kreft, M; Kunze, M; Schoetzau, A; Dölzlmüller, E; Eppel, W; Husslein, P; Ochsenbein-Koelble, N; Zimmermann, R; Bäz, E; Prömpeler, H; Bruder, E; Hahn, S; Hoesli, I

2016-10-12

As the accurate diagnosis and treatment of gestational diabetes mellitus (GDM) is of increasing importance; new diagnostic approaches for the assessment of GDM in early pregnancy were recently suggested. We evaluate the diagnostic power of an 'early' oral glucose tolerance test (OGTT) 75 g and glycosylated fibronectin (glyFn) for GDM screening in a normal cohort. In a prospective cohort study, 748 singleton pregnancies are recruited in 6 centres in Switzerland, Austria and Germany. Women are screened for pre-existing diabetes mellitus and GDM by an 'early' OGTT 75 g and/or the new biomarker, glyFn, at 12-15 weeks of gestation. Different screening strategies are compared to evaluate the impact on detection of GDM by an OGTT 75 g at 24-28 weeks of gestation as recommended by the International Association of Diabetes and Pregnancy Study Groups (IADPSG). A new screening algorithm is created by using multivariable risk estimation based on 'early' OGTT 75 g and/or glyFn results, incorporating maternal risk factors. Recruitment began in May 2014. This study received ethical approval from the ethics committees in Basel, Zurich, Vienna, Salzburg and Freiburg. It was registered under http://www.ClinicalTrials.gov (NCT02035059) on 12 January 2014. Data will be presented at international conferences and published in peer-reviewed journals. NCT02035059. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Evaluation of the impact of fetal fibronectin test implementation on hospital admissions for preterm labour in Ontario: a multiple baseline time-series design.

PubMed

Fell, D B; Sprague, A E; Grimshaw, J M; Yasseen, A S; Coyle, D; Dunn, S I; Perkins, S L; Peterson, W E; Johnson, M; Bunting, P S; Walker, M C

2014-03-01

To determine the impact of a health system-wide fetal fibronectin (fFN) testing programme on the rates of hospital admission for preterm labour (PTL). Multiple baseline time-series design. Canadian province of Ontario. A retrospective population-based cohort of antepartum and delivered obstetrical admissions in all Ontario hospitals between 1 April 2002 and 31 March 2010. International Classification of Diseases codes in a health system-wide hospital administrative database were used to identify the study population and define the outcome measure. An aggregate time series of monthly rates of hospital admissions for PTL was analysed using segmented regression models after aligning the fFN test implementation date for each institution. Rate of obstetrical hospital admission for PTL. Estimated rates of hospital admission for PTL following fFN implementation were lower than predicted had pre-implementation trends prevailed. The reduction in the rate was modest, but statistically significant, when estimated at 12 months following fFN implementation (-0.96 hospital admissions for PTL per 100 preterm births; 95% confidence interval [CI], -1.02 to -0.90, P = 0.04). The statistically significant reduction was sustained at 24 and 36 months following implementation. Using a robust quasi-experimental study design to overcome confounding as a result of underlying secular trends or concurrent interventions, we found evidence of a small but statistically significant reduction in the health system-level rate of hospital admissions for PTL following implementation of fFN testing in a large Canadian province. © 2013 Royal College of Obstetricians and Gynaecologists.

Isolation and Identification of Proteins Secreted by Cells Cultured within Synthetic Hydrogel-Based Matrices

PubMed Central

2018-01-01

Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture. PMID:29552635

Isolation and Identification of Proteins Secreted by Cells Cultured within Synthetic Hydrogel-Based Matrices.

PubMed

Sawicki, Lisa A; Choe, Leila H; Wiley, Katherine L; Lee, Kelvin H; Kloxin, April M

2018-03-12

Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostaining and confocal imaging confirmed these results and provided visualization of protein organization within the synthetic matrices. Additionally, culture medium was collected from the encapsulated hMSCs, and a Luminex assay was performed to identify secreted soluble factors, including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α). Together, these methods provide a unique approach for studying dynamic reciprocity between cells and synthetic microenvironments and have the potential to provide new biological insights into cell responses during three-dimensional (3D) controlled cell culture.

Quantitative fibronectin to help decision-making in women with symptoms of preterm labour (QUIDS) part 1: Individual participant data meta-analysis and health economic analysis.

PubMed

Stock, Sarah J; Wotherspoon, Lisa M; Boyd, Kathleen A; Morris, Rachel K; Dorling, Jon; Jackson, Lesley; Chandiramani, Manju; David, Anna L; Khalil, Asma; Shennan, Andrew; Hodgetts Morton, Victoria; Lavender, Tina; Khan, Khalid; Harper-Clarke, Susan; Mol, Ben W; Riley, Richard D; Norrie, John; Norman, Jane E

2018-04-07

The aim of the QUIDS study is to develop a decision support tool for the management of women with symptoms and signs of preterm labour, based on a validated prognostic model using quantitative fetal fibronectin (qfFN) concentration, in combination with clinical risk factors. The study will evaluate the Rapid fFN 10Q System (Hologic, Marlborough, Massachusetts) which quantifies fFN in a vaginal swab. In part 1 of the study, we will develop and internally validate a prognostic model using an individual participant data (IPD) meta-analysis of existing studies containing women with symptoms of preterm labour alongside fFN measurements and pregnancy outcome. An economic analysis will be undertaken to assess potential cost-effectiveness of the qfFN prognostic model. The primary endpoint will be the ability of the prognostic model to rule out spontaneous preterm birth within 7 days. Six eligible studies were identified by systematic review of the literature and five agreed to provide their IPD (n=5 studies, 1783 women and 139 events of preterm delivery within 7 days of testing). The study is funded by the National Institute of Healthcare Research Health Technology Assessment (HTA 14/32/01). It has been approved by the West of Scotland Research Ethics Committee (16/WS/0068). CRD42015027590. Protocol version 2, date 1 November 2016. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA

PubMed Central

Fusco, William G.; Choudhary, Neelima R.; Stewart, Shelley M.; Alam, S. Munir; Sempowski, Gregory D.; Elkins, Christopher

2015-01-01

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrAI) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine. PMID:25897604

Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA.

PubMed

Fusco, William G; Choudhary, Neelima R; Stewart, Shelley M; Alam, S Munir; Sempowski, Gregory D; Elkins, Christopher; Leduc, Isabelle

2015-04-01

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

PubMed

Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

Repurposed drug screen identifies cardiac glycosides as inhibitors of TGF-β-induced cancer-associated fibroblast differentiation.

PubMed

Coleman, David T; Gray, Alana L; Stephens, Charles A; Scott, Matthew L; Cardelli, James A

2016-05-31

The tumor microenvironment, primarily composed of myofibroblasts, directly influences the progression of solid tumors. Through secretion of growth factors, extracellular matrix deposition, and contractile mechanotransduction, myofibroblasts, or cancer-associated fibroblasts (CAFs), support angiogenesis and cancer cell invasion and metastasis. The differentiation of fibroblasts to CAFs is primarily induced by TGF-β from cancer cells. To discover agents capable of blocking CAF differentiation, we developed a high content immunofluorescence-based assay to screen repurposed chemical libraries utilizing fibronectin expression as an initial CAF marker. Screening of the Prestwick chemical library and NIH Clinical Collection repurposed drug library, totaling over 1700 compounds, identified cardiac glycosides as particularly potent CAF blocking agents. Cardiac glycosides are traditionally used to regulate intracellular calcium by inhibiting the Na+/K+ ATPase to control cardiac contractility. Herein, we report that multiple cardiac glycoside compounds, including digoxin, are able to inhibit TGF-β-induced fibronectin expression at low nanomolar concentrations without undesirable cell toxicity. We found this inhibition to hold true for multiple fibroblast cell lines. Using real-time qPCR, we determined that digoxin prevented induction of multiple CAF markers. Furthermore, we report that digoxin is able to prevent TGF-β-induced fibroblast contraction of extracellular matrix, a major phenotypic consequence of CAF differentiation. Assessing the mechanism of inhibition, we found digoxin reduced SMAD promoter activity downstream of TGF-β, and we provide data that the effect is through inhibition of its known target, the Na+/K+ ATPase. These findings support a critical role for calcium signaling during CAF differentiation and highlight a novel, repurposable modality for cancer therapy.

Differential regulations of fibronectin and laminin in Smad2 activation in vascular endothelial cells in response to disturbed flow.

PubMed

Yang, Tung-Lin; Lee, Pei-Ling; Lee, Ding-Yu; Wang, Wei-Li; Wei, Shu-Yi; Lee, Chih-I; Chiu, Jeng-Jiann

2018-01-02

Atherosclerosis occurs in arterial curvatures and branches, where the flow is disturbed with low and oscillatory shear stress (OSS). The remodeling and alterations of extracellular matrices (ECMs) and their composition is the critical step in atherogenesis. In this study, we investigated the effects of different ECM proteins on the regulation of mechanotransduction in vascular endothelial cells (ECs) in response to OSS. Through the experiments ranging from in vitro cell culture studies on effects of OSS on molecular signaling to in vivo examinations on clinical specimens from patients with coronary artery disease (CAD), we elucidated the roles of integrins and different ECMs, i.e., fibronectin (FN) and laminin (LM), in transforming growth factor (TGF)-β receptor (TβR)-mediated Smad2 activation and nuclear factor-κB (NF-κB) signaling in ECs in response to OSS and hence atherogenesis. OSS at 0.5±12 dynes/cm 2 induces sustained increases in the association of types I and II TβRs with β1 and β3 integrins in ECs grown on FN, but it only transient increases in ECs grown on LM. OSS induces a sustained activation of Smad2 in ECs on FN, but only a transient activation of Smad2 in ECs on LM. OSS-activation of Smad2 in ECs on FN regulates downstream NF-κB signaling and pro-inflammatory gene expression through the activation of β1 integrin and its association with TβRs. In contrast, OSS induces transient activations of β1 and β3 integrins in ECs on LM, which associate with type I TβR to regulate Smad2 phosphorylation, resulting in transient induction of NF-κB and pro-inflammatory gene expression. In vivo investigations on diseased human coronary arteries from CAD patients revealed that Smad2 is highly activated in ECs of atherosclerotic lesions, which is accompanied by the concomitant increase of FN rather than LM in the EC layer and neointimal region of atherosclerotic lesions. Our findings provide new insights into the mechanisms of how OSS regulates Smad2 signaling and pro-inflammatory genes through the complex signaling networks of integrins, TβRs, and ECMs, thus illustrating the molecular basis of regional pro-inflammatory activation within disturbed flow regions in the arterial tree.

Mono vs multilayer fibronectin coatings on polar/hydrophobic/ionic polyurethanes: Altering surface interactions with human monocytes.

PubMed

Gossart, Audrey; Battiston, Kyle G; Gand, Adeline; Pauthe, Emmanuel; Santerre, J Paul

2018-01-15

Monocyte interactions with materials that are biofunctionalized with fibronectin (Fn) are of interest because of the documented literature which associates this protein with white blood cell function at implant sites. A degradable-polar hydrophobic ionic polyurethane (D-PHI), has been reported to promote an anti-inflammatory response from human monocytes. The aim of the current work was to study the influence of intrinsic D-PHI material chemistry on Fn adsorption (mono and multi-layer structures), and to investigate the influence of such chemistry on the structural state of the Fn, as well as the latter's influence on the activity of human monocytes on the protein coated substrates. Significant differences in Fn adsorption, surface hydrophobicity and the availability of defined peptide sequences (N terminal, C terminal or Cell Binding Domain) for the Fn in mono vs multilayer structures were observed as a function of the changes in intrinsic material chemistry. A D-PHI-formulated polyurethane substrate with subtle changes in anionic and hydrophobic domain content relative to the polar non-ionic urethane/carbonate groups within the polymer matrix promoted the lowest activation of monocytes, in the presence of multi-layer Fn constructs. These results highlight the importance of chemical heterogeneity as a design parameter for biomaterial surfaces, and establishes a desired strategy for controlling human monocyte activity at the surface of devices, when these are coated with multi-layer Fn structures. The latter is an important step towards functionalizing the materials with multi-layer protein drug carriers as interventional therapeutic agents. The control of the behavior of monocytes, especially migration and activation, is of crucial interest to modulate the inflammatory response at the site of implanted biomaterial. Several studies report the influence of adsorbed serum proteins on the behavior of monocytes on biomaterials. However, few studies show the influence of surface chemical group distribution on the controlled adsorption and the subsequent induced conformation- of mono versus multi-layer assembled structures generated from specific proteins implicated in wound repair. The current research considered the role of Fn adsorption and conformation in thin films while interacting with the intrinsic chemistry of segmented block polyurethanes; and the influence of the former on modulation and activation of human monocytes. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Treating fructose-induced metabolic changes in mice with high-intensity interval training: insights in the liver, white adipose tissue, and skeletal muscle.

PubMed

Motta, Victor F; Bargut, Thereza L; Aguila, Marcia B; Mandarim-de-Lacerda, Carlos A

2017-10-01

Fructose-rich caloric sweeteners induce adverse changes in the metabolism of humans. The study evaluated the effects of high-intensity interval training (HIIT) on a fructose feeding model, focusing on the liver, white adipose tissue (WAT), skeletal muscle, and their interplay. Male C57BL/6 mice were fed for 18 wk one of the following diets: control (C; 5% of total energy from fructose) or fructose (F; 55% of total energy from fructose). In the 10th week, for an additional 8-wk period, the groups were divided into nontrained (NT) or HIIT groups, totaling four groups: C-NT, C-HIIT, F-NT, and F-HIIT. At the end of the experiment, fructose consumption in the F-NT group led to a high systolic blood pressure, high plasma triglycerides, insulin resistance with glucose intolerance, and lower insulin sensitivity. We also observed liver steatosis, adipocyte hypertrophy, and diminished gene expressions of peroxisome proliferator-activated receptor-γ coactivator 1-α and fibronectin type III domain containing 5 (FNDC5; irisin) in this F-NT group. These results were accompanied by decreased gene expressions of nuclear respiratory factor 1 and mitochondrial transcription factor A (markers of mitochondrial biogenesis), and peroxisome proliferator-activated receptor-α and carnitine palmitoyltransferase 1 (markers of β-oxidation). HIIT improved all of these data in the C-HIIT and F-HIIT groups. In conclusion, in mice fed a fructose diet, HIIT improved body mass, blood pressure, glucose metabolism, and plasma triglycerides. Liver, WAT, and skeletal muscle were positively modulated by HIIT, indicating HIIT as a coadjutant treatment for diseases affecting these tissues. NEW & NOTEWORTHY We investigated the effects of high-intensity interval training (HIIT) in mice fed a fructose-rich diet and the resulting severe negative effect on the liver, white adipose tissue (WAT), and skeletal muscle, which reduced the expression of fibronectin type III domain containing 5 (FNDC5, irisin) and PGC1α and, consequently, affected markers of mitochondrial biogenesis and β-oxidation. Because HIIT may block these adverse effects in all of these three tissues, it might be suggested that it functions as a coadjutant treatment in combatting the alterations caused by high-fructose intake. Copyright © 2017 the American Physiological Society.

Persistent Transmissible Gastroenteritis Virus Infection Enhances Enterotoxigenic Escherichia coli K88 Adhesion by Promoting Epithelial-Mesenchymal Transition in Intestinal Epithelial Cells.

PubMed

Xia, Lu; Dai, Lei; Yu, Qinghua; Yang, Qian

2017-11-01

Transmissible gastroenteritis virus (TGEV) is a coronavirus characterized by diarrhea and high morbidity rates, and the mortality rate is 100% in piglets less than 2 weeks old. Pigs infected with TGEV often suffer secondary infection by other pathogens, which aggravates the severity of diarrhea, but the mechanisms remain unknown. Here, we hypothesized that persistent TGEV infection stimulates the epithelial-mesenchymal transition (EMT), and thus enterotoxigenic Escherichia coli (ETEC) can more easily adhere to generating cells. Intestinal epithelial cells are the primary targets of TGEV and ETEC infections. We found that TGEV can persistently infect porcine intestinal columnar epithelial cells (IPEC-J2) and cause EMT, consistent with multiple changes in key cell characteristics. Infected cells display fibroblast-like shapes; exhibit increases in levels of mesenchymal markers with a corresponding loss of epithelial markers; have enhanced expression levels of interleukin-1β (IL-1β), IL-6, IL-8, transforming growth factor β (TGF-β), and tumor necrosis factor alpha (TNF-α) mRNAs; and demonstrate increases in migratory and invasive behaviors. Additional experiments showed that the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways via TGF-β is critical for the TGEV-mediated EMT process. Cellular uptake is also modified in cells that have undergone EMT. TGEV-infected cells have higher levels of integrin α5 and fibronectin and exhibit enhanced ETEC K88 adhesion. Reversal of EMT reduces ETEC K88 adhesion and inhibits the expression of integrin α5 and fibronectin. Overall, these results suggest that TGEV infection induces EMT in IPEC-J2 cells, increasing the adhesion of ETEC K88 in the intestine and facilitating dual infection. IMPORTANCE Transmissible gastroenteritis virus (TGEV) causes pig diarrhea and is often followed by secondary infection by other pathogens. In this study, we showed that persistent TGEV infection induces an EMT in porcine intestinal columnar epithelial cells (IPEC-J2) and enhances the adhesion of the secondary pathogen ETEC K88. Additional experiments suggest that integrin α5 and fibronectin play an important role in TGEV-enhanced ETEC K88 adhesion. Reversal of EMT reduces the expression of integrin α5 and fibronectin and also reduces ETEC K88 adhesion. We conclude that TGEV infection triggers EMT and facilitates dual infection. Our results provide new insights into secondary infection and suggest that targeted anti-EMT therapy may have implications for the prevention and treatment of secondary infection. Copyright © 2017 American Society for Microbiology.

Fetal fibronectin, cervical length, and the risk of preterm birth in patients with an ultrasound or physical exam indicated cervical cerclage.

PubMed

Kim, Renita S; Gupta, Simi; Lam-Rachlin, Jennifer; Saltzman, Daniel H; Rebarber, Andrei; Fox, Nathan S

2016-11-01

The objective of this study is to estimate the risk of preterm birth in patients with an ultrasound or physical exam indicated cervical cerclage based on the results of fetal fibronectin (fFN) and cervical length (CL) screening. Retrospective cohort of patients with a singleton pregnancy and an ultrasound or physical exam indicated Shirodkar cerclage placed by one maternal-fetal medicine practice from November 2005 to January 2015. Patients routinely underwent serial CL and fFN testing from 22 to 32 weeks. Based on ROC curve analysis, a short CL was defined as ≤15 mm. All fFN and CL results included are from after the cerclage placement. One hundred and four patients were included. Seventy eight (75%) patients had an ultrasound-indicated cerclage and 26 (25%) patients had a physical exam-indicated cerclage. A positive fFN was associate with preterm birth <32 weeks (15.6% versus 4.2%, p = 0.043),  <35 weeks (37.5% versus 11.1%, p = 0.002),  <37 weeks (65.6% versus 20.8%, p < 0.001), and earlier gestational ages at delivery (35.2 ± 3.9 versus 37.4 ± 2.9, p = 0.001). A short CL was also associated with preterm birth <35 weeks (50.0% versus 11.9%, p < 0.01), preterm birth <37 weeks (55.0% versus 29.8%, p = 0.033), and earlier gestational ages at delivery (34.8 ± 4.1 versus 37.2 ± 3.0, p = 0.004). The risk of preterm birth <32,  <35, and <37 weeks increased significantly with the number of abnormal markers. In patients with an ultrasound or physical exam indicated cerclage, a positive fFN and a short CL are both associated with preterm birth. The risk of preterm birth increases with the number of abnormal biomarkers.

Rosiglitazone reduces renal and plasma markers of oxidative injury and reverses urinary metabolite abnormalities in the amelioration of diabetic nephropathy.

PubMed

Zhang, Hongyu; Saha, Jharna; Byun, Jaeman; Schin, MaryLee; Lorenz, Matthew; Kennedy, Robert T; Kretzler, Matthias; Feldman, Eva L; Pennathur, Subramaniam; Brosius, Frank C

2008-10-01

Recent studies suggest that thiazolidinediones ameliorate diabetic nephropathy (DN) independently of their effect on hyperglycemia. In the current study, we confirm and extend these findings by showing that rosiglitazone treatment prevented the development of DN and reversed multiple markers of oxidative injury in DBA/2J mice made diabetic by low-dose streptozotocin. These diabetic mice developed a 14.2-fold increase in albuminuria and a 53% expansion of renal glomerular extracellular matrix after 12 wk of diabetes. These changes were largely abrogated by administration of rosiglitazone beginning 2 wk after the completion of streptozotocin injections. Rosiglitazone had no effect on glycemic control. Rosiglitazone had similar effects on insulin-treated diabetic mice after 24 wk of diabetes. Podocyte loss and glomerular fibronectin accumulation, other markers of early DN, were prevented by rosiglitazone in both 12- and 24-wk diabetic models. Surprisingly, glomerular GLUT1 levels did not increase and nephrin levels did not decrease in the diabetic animals; neither changed with rosiglitazone. Plasma and kidney markers of protein oxidation and lipid peroxidation were significantly elevated in the 24-wk diabetic animals despite insulin treatment and were reduced to near-normal levels by rosiglitazone. Finally, urinary metabolites were markedly altered by diabetes. Of 1,988 metabolite features identified by electrospray ionization time of flight mass spectrometry, levels of 56 were altered more than twofold in the urine of diabetic mice. Of these, 21 were returned to normal by rosiglitazone. Thus rosiglitazone has direct effects on the renal glomerulus to reduce reactive oxygen species accumulation to prevent type 1 diabetic mice from development of DN.

Integrin expression and glycosylation patterns regulate cell-matrix adhesion and alter with breast cancer progression.

PubMed

Singh, Chandrajeet; Shyanti, Ritis K; Singh, Virendra; Kale, Raosaheb K; Mishra, Jai P N; Singh, Rana P

2018-05-05

Integrins are the major cell adhesion glycoproteins involved in cell-extracellular matrix (ECM) interaction and metastasis. Further, glycosylation on integrin is necessary for its proper folding and functionality. Herein, differential expression of integrins viz., αvβ3 and αvβ6 was examined in MDA-MB-231, MDA-MB-468 and MCF-10A cells, which signify three different stages of breast cancer development from highly metastatic to non-tumorigenic stage. The expression of αvβ3 and αvβ6 integrins at mRNA and protein levels was observed in all three cell lines and the results displayed a distinct pattern of expression. Highly metastatic cells showed enhanced expression of αvβ3 than moderate metastatic and non-tumorigenic cells. The scenario was reversed in case of αvβ6 integrin, which was strongly expressed in moderate metastatic and non-tumorigenic cells. N-glycosylation of αvβ3 and αvβ6 integrins is required for the attachment of cells to ECM proteins like fibronectin. The cell adhesion properties were found to be different in these cancer cells with respect to the type of integrins expressed. The results testify that αvβ3 integrin in highly metastatic cells, αvβ6 integrin in both moderate metastatic and non-tumorigenic cells play an important role in cell adhesion. The investigation typify that N-glycosylation on integrins is also necessary for cell-ECM interaction. Further, glycosylation inhibition by Swainsonine is found to be more detrimental to invasive property of moderate metastatic cells. Conclusively, types of integrins expressed as well as their N-glycosylation pattern alter during the course of breast cancer progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Esculetin ameliorates hepatic fibrosis in high fat diet induced non-alcoholic fatty liver disease by regulation of FoxO1 mediated pathway.

PubMed

Pandey, Anuradha; Raj, Priyank; Goru, Santosh Kumar; Kadakol, Almesh; Malek, Vajir; Sharma, Nisha; Gaikwad, Anil Bhanudas

2017-08-01

Non-alcoholic fatty liver disease (NAFLD), a chronic metabolic disorder is associated with oxidative stress, inflammation and fibrotic cascades. In this study, we aimed to examine the effects of Esculetin, a well-known anti-oxidant on TGF-β1 mediated liver fibrosis and FoxO1 activity. A non-genetic murine model for NAFLD was developed by chronic high fat diet (HFD) (58% calories from fats) feeding in Wistar rats. The plasma biochemical parameters, liver function tests, oxidative stress, and histopathological alterations were assessed. The alterations in extracellular matrix (ECM) deposition and FoxO1 activity were assessed by immunohistochemistry. The aberrations in plasma parameters, liver functioning, morphometric and microscopic changes in liver structure of HFD fed rats were significantly improved by treatment with Esculetin. Liver fibrosis, identified in the form of collagen deposition and expression of fibrotic proteins like TGF-β1 and fibronectin was also markedly controlled by Esculetin. The expression of phospho-FoxO1 was found to be reduced in HFD fed rats' liver, showing an increase in activation of FoxO1 under insulin resistant and hyperglycemic states. Esculetin treatment could improve phospho-FoxO1 expression, thus showing its ability to act on Akt/PI3K/FoxO1 pathway. As per the previous studies, a potential therapy for NAFLD may be the one with multi-faceted actions on insulin resistance, oxidative stress, inflammation and fibrosis. This study demonstrates the efficiency of Esculetin in improving liver fibrosis in HFD induced NAFLD. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Gene Expression Profiling in Limb-Girdle Muscular Dystrophy 2A

PubMed Central

Sáenz, Amets; Azpitarte, Margarita; Armañanzas, Rubén; Leturcq, France; Alzualde, Ainhoa; Inza, Iñaki; García-Bragado, Federico; De la Herran, Gaspar; Corcuera, Julián; Cabello, Ana; Navarro, Carmen; De la Torre, Carolina; Gallardo, Eduard; Illa, Isabel; de Munain, Adolfo López

2008-01-01

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that β-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1). Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies. PMID:19015733

Space shuttle flight (STS-45) of L8 myoblast cells results in the isolation of a nonfusing cell line variant.

PubMed

Kulesh, D A; Anderson, L H; Wilson, B; Otis, E J; Elgin, D M; Barker, M J; Mehm, W J; Kearney, G P

1994-08-01

Myoblast cell cultures have been widely employed in conventional (1g) studies of biological processes because characteristics of intact muscle can be readily observed in these cultured cells. We decided to investigate the effects of spaceflight on muscle by utilizing a well characterized myoblast cell line (L8 rat myoblasts) as cultured in the recently designed Space Tissue Loss Flight Module "A" (STL-A). The STL-A is a "state of the art," compact, fully contained, automated cell culture apparatus which replaces a single mid-deck locker on the Space Shuttle. The L8 cells were successfully flown in the STL-A on the Space Shuttle STS-45 mission. Upon return to earth, reculturing of these spaceflown L8 cells (L8SF) resulted in their unexpected failure to fuse and differentiate into myotubes. This inability of the L8SF cells to fuse was found to be a permanent phenotypic alteration. Scanning electron microscopic examination of L8SF cells growing at 1g on fibronectin-coated polypropylene fibers exhibited a strikingly different morphology as compared to control cells. In addition to their failure to fuse into myotubes, L8SF cells also piled up on top of each other. When assayed in fusion-promoting soft agar, L8SF cells gave rise to substantially more and larger colonies than did either preflight (L8AT) or ground control (L8GC) cells. All data to this point indicate that flying L8 rat myoblasts on the Space Shuttle for a duration of 7-10 d at subconfluent densities results in several permanent phenotypic alterations in these cells.

Effects of Phonation Time and Magnitude Dose on Vocal Fold Epithelial Genes, Barrier Integrity, and Function

PubMed Central

Kojima, Tsuyoshi; Valenzuela, Carla V.; Novaleski, Carolyn K.; Van Deusen, Mark; Mitchell, Joshua R.; Garrett, C. Gaelyn; Sivasankar, M. Preeti; Rousseau, Bernard

2014-01-01

Objective To investigate the effects of increasing time and magnitude doses of vibration exposure on transcription of the vocal fold's junctional proteins, structural alterations, and functional tissue outcomes. Study Design Animal study. Methods 100 New Zealand White breeder rabbits were studied. Dependent variables were measured in response to increasing time doses (30, 60, or 120 minutes) and magnitude doses (control, modal intensity, and raised intensity) of vibration exposure. Messenger RNA expression of occludin, zonula occluden-1 (ZO-1), E-cadherin, β-catenin, interleukin 1β (IL-1β), cyclooxygenase-2 (COX-2), transforming growth factor β-1 (TGFβ1), and fibronectin were measured. Tissue structural alterations were assessed using transmission electron microscopy (TEM). Transepithelial resistance was used to measure functional tissue outcomes. Results Occludin gene expression was downregulated in vocal folds exposed to 120 minute time doses of raised intensity phonation, relative to control, and modal intensity phonation. ZO-1 gene expression was upregulated following a 120 minute time dose of modal intensity phonation, compared to control, and downregulated after a 120 minute time dose of raised intensity phonation, compared to modal intensity phonation. E-cadherin gene expression was downregulated after a120 minute time dose of raised intensity phonation, compared to control and modal intensity phonation. TEM revealed extensive desquamation of the stratified squamous epithelial cells with increasing time and magnitude doses of vibration exposure. A general observation of lower transepithelial resistance measures was made in tissues exposed to raised intensity phonation, compared to all other groups. Conclusions This study provides evidence of vocal fold tissue responses to varying time and magnitude doses of vibration exposure. Level of Evidence N/A PMID:25073715

Tropomyosin isoform Tpm2.1 regulates collective and amoeboid cell migration and cell aggregation in breast epithelial cells.

PubMed

Shin, HyeRim; Kim, Dayoung; Helfman, David M

2017-11-10

Metastasis dissemination is the result of various processes including cell migration and cell aggregation. These processes involve alterations in the expression and organization of cytoskeletal and adhesion proteins in tumor cells. Alterations in actin filaments and their binding partners are known to be key players in metastasis. Downregulation of specific tropomyosin (Tpm) isoforms is a common characteristic of transformed cells. In this study, we examined the role of Tpm2.1 in non-transformed MCF10A breast epithelial cells in cell migration and cell aggregation, because this isoform is downregulated in primary and metastatic breast cancer as well as various breast cancer cell lines. Downregulation of Tpm2.1 using siRNA or shRNA resulted in retardation of collective cell migration but increase in single cell migration and invasion. Loss of Tpm2.1 is associated with enhanced actomyosin contractility and increased expression of E-cadherin and β-catenin. Furthermore, inhibition of Rho-associated kinase (ROCK) recovered collective cell migration in Tpm2.1-silenced cells. We also found that Tpm2.1-silenced cells formed more compacted spheroids and exhibited faster cell motility when spheroids were re-plated on 2D surfaces coated with fibronectin and collagen. When Tpm2.1 was downregulated, we observed a decrease in the level of AXL receptor tyrosine kinase, which may explain the increased levels of E-cadherin and β-catenin. These studies demonstrate that Tpm2.1 functions as an important regulator of cell migration and cell aggregation in breast epithelial cells. These findings suggest that downregulation of Tpm2.1 may play a critical role during tumor progression by facilitating the metastatic potential of tumor cells.

Hypergravity Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

NASA Technical Reports Server (NTRS)

Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

2003-01-01

Extensive characterizations of the physiologic consequences of microgravity and gravity indicate that lack of weight-bearing may cause tissue atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) in mechanosensitive tissues. Recent work from our laboratory and from others shows that an increase of gravity increases bone cell growth and survival. We found that 50-g hypergravity stimulation increased osteoblast proliferation for cells grown on Collagen Type I and Fibronectin, but not on Laminin or uncoated plastic. This may be a tissue-specific response, because 50-g hypergravity stimulation caused no increase in proliferation for primary rat fibroblasts. These results combined with RT-PCR for all possible integrins indicate that beta1 integrin subunit may be involved. The osteoblast proliferation response on Collagen Type I was greater at 25-g than at 10-g or 50-g; 24-h duration of hypergravity was necessary to see an increase in proliferation. Survival was enhanced during hypergravity stimulation by the presence of matrix. Flow cytometry analysis indicated that cell cycle may be altered; BrdU incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. To further investigate the molecular components involved, we applied fluorescence labeling of cytoskeletal and signaling molecules to cells after 2 to 30 minutes of hypergravity stimulation. While structural components did not appear to be altered, phosphorylation increased, indicating that signaling pathways may be activated. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signaling pathways which are sensitive to duration and g-level.