Involvement Of Fibronectin Research Articles

Involvement of Fibronectin and Matrix Metalloproteinases in Airway Smooth Muscle Cell Migration for the Process of Airway Remodeling

Airway remodeling is a repair process occurring after airway injury; its primary histopathological features are subepithelial fibrosis and smooth muscle thickening of the bronchi. These histopathological changes are considered to occur due to bronchial smooth muscle cells (bSMC) that secrete extracellular matrix (ECM) proteins, which work as chemoattractants and influence cell migration. Therefore, we examined the interaction between bSMCs and ECM proteins in vitro for understanding the remodeling process in the bronchi. bSMCs were cultured to collect a bSMC-conditioned medium. Using the bSMC-conditioned medium thus obtained, we performed a cell migration assay, characterized beta integrin expression, and identified ECM proteins and matrix metalloproteinases by western blotting and gelatin zymography, respectively. The response of bSMC migration to bSMC-conditioned medium increased with time in culture, and fibronectin (FIB) was detected as a chemoattractant for bSMCs in bSMC-conditioned medium by western blot analysis and a cell migration assay using anti-FIB antibodies. The involvement of beta1 integrin in the migration of bSMCs toward FIB contained in bSMC-conditioned medium was demonstrated by inhibition of cell migration using anti-beta1 integrin antibodies. Expression of beta1 integrin on bSMCs was confirmed by using a beta-integrin-mediated cell adhesion array. In addition, metalloproteinases detected in bSMC-conditioned medium by gelatin zymography were suggested to be matrix metalloproteinase-1 and 2 by western blotting and amino acid sequencing. Our results suggest that FIB and matrix metalloproteinases secreted from bSMCs might play major roles in bSMC migration in the process of airway remodeling.

Chromosomalgfplabelling ofPseudomonas aeruginosausing a mini-Tn7transposon: application for studies of bacteria–host interactions

Analysis of bacterial interactions with host cells using multiple techniques is essential for studies on microbial pathogenesis and for the development of new antimicrobial therapies. Pseudomonas aeruginosa is an important opportunistic pathogen that can cause severe, often life-threatening pulmonary infections in individuals with impaired host defense mechanisms. Using a mini-Tn7 transposon delivery system, we have chromosomally labelled the strain P. aeruginosa PAK with a green fluorescent protein gene (gfp) and tested PAKgfp as a research tool for studies of bacteria-host interactions. We were able to reliably and rapidly measure the interactions of PAKgfp with A549 human lung epithelial cells by using flow cytometry, a fluorometric microplate reader-based assay, and fluorescence microscopy. With these analytical tools, we have demonstrated the adhesion of PAKgfp to the extracellular matrix protein fibronectin and the involvement of fibronectin in PAKgfp-A549 cell interactions. PAKgfp can be successfully used to explore the effects of various pharmacological compounds on P. aeruginosa - host cell interactions in both in vitro and in vivo systems, with potentially important medical applications.

Association study between fibronectin and coronary heart disease

Fibronectin is a known chemoattractant for several cell types that play a role in the wound healing process, including fibroblasts, endothelial cells and macrophages. It also generates a scaffold that allows attachment of other extracellular matrix components. Large amounts of fibronectin have been detected in atherosclerotic plaques, suggesting that it may play a role in the pathogenesis of atherosclerosis. To examine the possible involvement of fibronectin in the etiology of atherosclerotic coronary heart disease, we analyzed four polymorphisms in the human fibronectin gene and determined the plasma fibronectin levels in patients with coronary heart disease (n = 109) and age- and gender-matched controls (n = 123) in Chinese Han people. No significant positive association was observed between these polymorphisms and coronary heart disease. The levels of circulating plasma fibronectin, however, were significantly lower in patients with coronary heart disease (mean +/- SD 245 +/- 87 mg/L) compared with controls (354 +/- 88 mg/L) (p < 0.001). The odds ratio (OR) for plasma fibronectin was 0.94 in a multivariate unconditional logistic regression model (OR = 0.94, 95% CI 0.91-0.96, p < 0.001). We conclude that, in our population, the four fibronectin gene polymorphisms detected are not associated with clinical coronary heart disease. Our data suggest that low circulating fibronectin levels might be a new marker of coronary heart disease.

Association study between the fibronectin gene and schizophrenia.

Fibronectin is one of the cell adhesion proteins. Adhesion molecules play an important role in neural and synaptic genesis, and their dysfunction may result in neurodevelopmental abnormalities, which have been assumed to be a factor in the pathogenesis of schizophrenia. To examine the possible involvement of fibronectin in the etiology of schizophrenia, we analyzed six polymorphisms, located in introns 2, 21, 24, and 26, and exons 20 and 28, in the human fibronectin gene (FN1) of schizophrenic patients in the Japanese population (n = 104) and age-and gender-matched controls (n = 104). No significant positive association was observed between either of the polymorphisms and schizophrenia, nor was an association found between either of the polymorphisms and any subtypes of schizophrenia. These data did not provide evidence for a contribution of the FN1 gene to susceptibility to schizophrenia.

The interaction of cellular fibronectin with collagen during fibroblast-mediated contraction of collagen gels.

In the first instance highly hydrated collagen gels contract to dense and compact gels when populated by fibroblasts. We previously reported the involvement of fibronectin (FN) in an early process of the collagen gel contraction, utilizing a specific monoclonal antibody dubbed A3A5 (MoAb-A3A5) that inhibits the gel contraction. This study was performed to further characterize the role of the epitope for MoAb-A3A5 in the interaction between fibroblasts and collagen fibrils. Although both cellular FN (cFN) and plasma FN (pFN) were reactive with MoAb-A3A5, the FN that actually participates in a process of the gel contraction was shown to be cFN. The gel contraction was significantly accelerated when fibroblasts were pretreated with excess amounts of cFN and was significantly inhibited when the collagen molecules were pretreated with excess cFN. Such effects of the pretreatments were not observed for pFN. The involvement of cFN, but not pFN, in the interaction of fibroblasts with collagen fibrils was additionally shown by the similar inhibitory action of cFN, but not pFN, on the spreading and elongation of fibroblasts on collagen fibrils. The epitope for MoAb-A3A5 was strongly suggested to be a new functional domain responsible for the interactions between fibroblasts and native collagen molecules. This was not the case for those with denatured one, because fibroblasts on collagen fibrils were not stainable with MoAb-A3A5, whereas the interactions on gelatin were stainable. The lack of the reactivity of fibroblasts on collagen fibrils toward MoAb-A3A5 was not a result of the absence of FN on the cell membrane, but seemed to be a steric hindrance to the access of the antibody.

Involvement of fibronectin in the regulation of urokinase production and binding in murine mammary tumor cells

Involvement of fibronectin in the regulation of urokinase production and binding in murine mammary tumor cells

Involvement of fibronectin in the regulation of urokinase production and binding in murine mammary tumor cells.

Tumor invasion and metastasis development is a multistep process involving adhesion molecules as well as tumor proteases. It has been reported that tumor cells lacking fibronectin (FN) expression and engineered to re-express FN showed a marked reduction in metastatic ability. Besides its effects on cell adhesion and migration, FN could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we analyzed the production of urokinase-type plasminogen activator (uPA), and its receptor (uPAR), 2 molecules involved in the invasive phenotype, in cells over-expressing RGD wild-type FN (FNwt clones) or RGD-mutated FN (FN RGD-minus clones). Secreted uPA activity and antigen were significantly up-regulated in FN-expressing clones, although RGD-minus cells secreted approximately 50% less uPA than the FNwt ones. Interestingly, while control and FN RGD-minus clones were able to readily bind uPA to their surface, FNwt clones exhibited impaired uPA binding. Furthermore, treatment of the parental cell line as well as the control and FN-expressing clones with exogenous purified FN or RGD peptides induced up-regulation of uPA production and the reduction of uPA membrane binding, which was associated with lower expression of uPAR. This modulation by FN was found to be dependent on RGD sequence and beta1 integrin. These results strongly suggest a novel activity for the multifunctional glycoprotein FN regarding the regulation of uPA production as well as the capacity of tumor cells to bind uPA.

Increased Expression of Fibronectin Isoforms After Myocardial Infarction in Rats

Fibronectin is a known chemoattractant for several cell types which play a role in the wound healing process, like fibroblasts, endothelial cells and macrophages. In addition, fibronectin generates a scaffold to which other extracellular matrix components can attach. The possible involvement of fibronectin in the wound healing process after myocardial infarction (MI) was investigated by studying the expression of fibronectin isoforms after induction of a MI in the rat. Deposition of plasma (pFN) and cellular fibronectin (cFN) protein was determined immunohistochemically, using monoclonal antibodies specific for cFN and polyclonal anti-human total FN (tFN antibodies). Expression of the mRNAs of total cFN and the embryonic isoforms EIIIA and EIIIB was investigated, usingin situhybridization (ISH). The ratio between EIIIA containing fibronectin (EIIIA+–FN) mRNA and total cFN mRNA was determined using a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). cFN protein was present from day 4 until day 35 after infarction and was located around the area of infarction, in the epi- and endomyocardium and in the wall of larger vessels. pFN was found in the infarcted cardiomyocytes 1 day after the induction of the MI. From day 4 on pFN protein deposition was found in the border zone of the infarction and in the wall of larger vessels. pFN immunoreactivity remained present at high levels around the area of infarction and in the vessel wall throughout the entire period of investigation (90 days). From day 35 after the infarction pFN protein was detected in cardiomyocytes of the right ventricle and septum. cFN mRNA, determined byin situhybridization, was present in the border zone of the infarcted area as early as 1 day after MI, and its expression peaked at 4 days after MI. Four days after MI the mRNA's coding for both the embryonic isoforms EIIIA and EIIIB could also be detected in the same area. Because expression of the EIIIA isoform was more abundant than the EIIIB isoform we only determined the percentage of the EIIIIA containing isoform from total FN. EIIIA+mRNA was elevated 1 day after MI. We conclude that various fibronectin isoforms including the embryonic isoforms accumulate in the heart after MI. This suggests that these isoforms may play a role in the wound healing process in the left ventricle of the infarcted heart.

Role of LFA-1, CD2, VLA-5/CD29, and CD43 Surface Receptors in CD4 + T Cell Adhesion to B Cells

We investigated the adhesion to B cells of CD4 + T cells both in the resting state and following activation by CD3 cross-linking or stimulation by PMA/ionomycine/IL2 for 6 days. Both resting and activated CD4 + T cell adhesion were inhibited by anti-LFA-1, -CD2, -VLA-5/CD29, and -CD43 antibodies, suggesting coordinated upregulation of T cell adhesion. The CD2 and LFA-1 adhesion pathways were found to act independently, as CD2 was functional in T cells not expressing LFA-1, and vice versa, and as specific antibodies had additive effects. In contrast, LFA-1- and VLA-5/CD29-specific antibodies did not have an additive blocking effect on CD4 + T cell adhesion, suggesting that efficient adhesion requires a competitive association of integrins with cytoskeleton elements. Although the involvement of fibronectin (coated to B cells via VLA-4) in VLA-5-mediated T cell adhesion to B cells is feasible, an anti-fibronectin and a VLA-4-specific antibody had no blocking effect. The involvement of an unidentified B cell ligand can also be envisaged.

Involvement of fibronectin and interacting serum components in the regulation of activity of human natural killer cells

The cytotoxic activity of natural killer cells and the intensity of conjugate formation are studiedin vitro in the natural cytotoxicity reaction against3H-uridine-labeled human erythromyeloleukotic cells K-562 in the presence of fibronectin, γ-globulin, and fibronectin/γ-globulin combination. It is demonstrated that fibronectin does not change natural cytotoxicity, γ-globulin increases the activity of human natural killer cells, and the fibronectin — γ-globulin combination increases both the intensity of conjugate formation and the cytotoxic activity of natural killer cells.

Microinjection of antifibronectin antibodies in the chicken blastoderm: Inhibition of mesoblast cell migration but not of cell ingression at the primitive streak

The involvement of fibronectin in adhesion and migration of individual mesoblast cells during chicken gastrulation was examined after microinjection of functional and nonfunctional antifibronectin antibodies in the blastoderm during the period of rapid migration of mesoblast cells. The injection of affinity-purified polyclonal antihuman fibronectin antibody (total IgG or Fab fragment) or of monoclonal antichicken cellular fibronectin caused a thickening of the primitive streak, which was composed of loosely connected cells. This effect was most evident at the level of Hensen's node, and very few mesoblast cells were observed migrating in the space between upper layer and deep layer. The obvious explanation of this effect was that the de-epithelialization of upper layer cells persisted in the presence of antibodies, but ingressed cells failed to emigrate from the primitive streak. Immunostaining of microinjected antibodies showed binding to the basement membrane, to the cell surface of mesoblast cells that had migrated before microinjection occurred, and to the cell surface of deep layer cells. Cells that ingressed and detached in the course of reincubation of the embryo possessed little immunolabelling along their cell surface. The results suggest that the failure of ingressed cells to emigrate from the primitive streak and to form mesoblast was due (1) to alterations in adhesion between newly ingressed primitive streak cells, which had the ability to detach but possessed relatively little fibronectin along their cell surfaces and a small number of cell protrusions, and (2) probably to a lack of adhesion of detached cells to the basement membrane, which was blocked by the presence of antifibronectin antibodies. We conclude that the presence of fibronectin in the basement membrane is required for emigration of ingressed cells and migration of mesoblast cells to occur. Once migration has commenced, fibronectin is also deposited along the cell surface of migrating cells, a factor that may increase their mutual adhesion.

Involvement of fibronectin during epiboly and gastrulation in embryos of the common carp, Cyprinus carpio.

The present report firstly describes a pilot study in which, during early development of embryos of the common carp, Cyprinus carpio, the cellular adhesion to fibronectin (FN) was blocked by administration of GRGDS peptide (which binds to the FN-receptor). As this treatment resulted in developmental aberrations, suggesting a functional role for FN, the major part of the work was focussed on the distribution of reactivity of anti-FN antibodies during epiboly and gastrulation. GRGDS treatment had a concentration dependent effect on development. Incubation of embryos in 1.5 mg/ml from the 32-cell stage onwards caused a retardation of epiboly, which did not proceed beyond 60%. The embryos did not show involution, as was confirmed by histological study. These preliminary results suggest that FN is involved in both epiboly and gastrulation of carp embryos. During cleavage, no specific extracellular binding of anti-FN antiserum could be observed. However, binding to a number of cell membranes took place from early epiboly onwards. After the onset of gastrulation, we observed a gradually increasing number of the deepest epiblast cells, showing immunostaining on part of their surface, facing the yolk syncytial layer (YSL) or the involuted cells. During early epiboly, anti-FN binding was restricted to areas in front of the migratory hypoblast cells. Later on, binding was found at the border of hypoblast and epiblast cells. At 100% epiboly, some contact areas of epiblast and hypoblast showed a discontinuous lining of reactivity, whilst other areas appeared devoid of anti-FN binding sites. The results indicate that FN is involved in the migration and guidance of hypoblast cells during gastrulation in carp.

Involvement of Fibronectin and Its Receptor in Human Lymphocyte Proliferation

Adhesion between lymphocytes and antigen-presenting cells is necessary for the development of certain immune reactions. We have previously shown that fibronectin (FN) added to mixed lymphocyte cultures (MLC) can restore a decreased lymphocyte proliferation in immunocompromised individuals. Using highly purified cell populations from peripheral blood for depletion and adding back experiments we show here that exogenous FN enhanced proliferation only when allogeneic monocytes were co-cultured with responder lymphocytes. Although lymphocyte proliferation in MLC was augmented by FN, there was no preferential proliferation of any particular major lymphocyte subpopulation in cultures supplemented with FN as compared to control cultures lacking its addition. Antibody against the FN receptor (FN-R) of the beta 1 integrin family, as well as Arg-Gly-Asp containing peptide, could inhibit alloantigen-induced lymphocyte proliferation in a concentration-dependent manner. Anti-CD3-induced proliferation was inhibited by anti-FN-R antibody but not Arg-Gly-Asp peptide whereas no inhibition was seen with the phytohemagglutinin (PHA)-induced lymphocyte proliferation. This study presents further evidence that FN and its receptor (alpha 5 beta 1) are involved in the augmentation of T-cell responsiveness to proliferative stimuli.

Antibodies to different members of the β1 (CD29) integrins induce homotypic and heterotypic cellular aggregation

Members of the β 1 (CD29) integrin family are involved in cellular adhesion to extracellular matrix. However, there have been several reports of CD29 integrin participation in intercellular adhesion. For example, the treatment of the human T cell line Jurkat with antibodies to α 4 (CD49d) causes homotypic aggregation. The present report describes the induction of aggregation of Jurkat cells by antibodies to α 5 (CD49e) and to a lesser extent by antibodies to the common β 1 CD29 chain of these integrins. The metabolic requirements for these aggregations are compared with that of the CD49d-induced process. The possible involvement of fibronectin in the cytoadhesion appears to be unlikely as (1) the aggregates form in the absence of plasma fibronectin; (2) antibodies to fibronectin do not inhibit the cell adhesion; and (3) exogenous fibronectin does not influence the process. One of the cognate partners involved in the CD49e-induced aggregation appears to be present on non-antibody-treated Jurkat cells as the cells are coincorporated into aggregates of anti-CD49e-stimulated cells. The adhesion does not appear to involve members of the CD2, CD3, CD4, or CD18 receptor groups. These results indicate that interaction with α 4, α 5 chain or the β 1, chain of the CD29 integrins leads to the induction of intercellular adhesion. The possible biological significance of this process is discussed.

Interaction of Candida albicans with genital mucosal surfaces: involvement of fibronectin in adherence.

Interaction of Candida albicans with genital mucosal surfaces: involvement of fibronectin in adherence.

Involvement of fibronectin in in vitro regeneration of retinal pigment epithelium.

Argon laser photocoagulation was placed on the confluent monolayer of cultured chick retinal pigment epithelial cells as a model of the regeneration process of retinal pigment epithelium after laser burn. The intense fibrillar net immunofluorescent pattern of fibronectin appeared on the burnt area from 2 h after the laser application, before the beginning of tissue reconstruction. Fibronectin was observed for several days, then became undetectable before the complete regeneration of retinal pigment epithelial cells. This indicates that fibronectin is involved in the early regeneration process of retinal pigment epithelium.

Ganglioside inhibition of attachment and differentiation of cultured rat granulosa cells: interactions with fibronectin.

The involvement of fibronectin in the attachment and differentiation of rat granulosa cells, cultured in a chemically defined medium, was investigated using the inhibitory properties of mixed brain gangliosides (MBGs) and highly purified disialoganglioside, GD1a. MBGs inhibited cell attachment to plastic culture surfaces in a concentration-dependent manner, with 0.1 mmol l-1 causing significantly decreased attachment between 0.5 and 24 h of incubation. Inhibition of attachment to a fibronectin-coated substratum was even greater. The inhibitory effect of MBGs was not caused by binding to the cell surface, but instead the inhibitory factor(s) were adsorbed on a surface of immobilized human plasma fibronectin, thereby preventing cell attachment to this surface. The inhibitory action of MBGs was also neutralized by the addition of soluble fibronectin. Furthermore, at least one component of MBGs, detected chemically following thin-layer chromatography, was directly shown to bind to human fibronectin. MBGs inhibited to varying degrees the follicle-stimulating hormone(FSH)-dependent responses: augmentation of cellular protein content, production of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and progestins (progesterone + 20 alpha-hydroxypregn-4-en-3-one + pregnenolone), and induction of aromatase activity. These inhibitory activities of MBGs could not be eliminated by adsorption on immobilized fibronectin or reversed by addition of soluble fibronectin, thus distinguishing these actions from the early inhibition of cell attachment. FSH-dependent responses were also inhibited by GD1a, while responses to stimulation by dibutyryl cyclic AMP plus 3-isobutyl-1-methyl xanthine were less affected by this ganglioside. These results suggest that gangliosides inhibit attachment of granulosa cells in culture by binding to fibronectin, whereas the inhibition of FSH-dependent differentiation occurs by other modes of action that are unrelated to the effects on cell adhesion.

Extracellular matrix synthesis during mesenchymal cell condensation and chondrogenesis: [formula omitted], Dept. of Anatomy, New Jersey Medical School, Newark, NJ., USA

Fibronectin is a known chemoattractant for several cell types which play a role in the wound healing process, like fibroblasts, endothelial cells and macrophages. In addition, fibronectin generates a scaffold to which other extracellular matrix components can attach. The possible involvement of fibronectin in the wound healing process after myocardial infarction (MI) was investigated by studying the expression of fibronectin isoforms after induction of a MI in the rat. Deposition of plasma (pFN) and cellular fibronectin (cFN) protein was determined immunohistochemically, using monoclonal antibodies specific for cFN and polyclonal anti-human total FN (tFN antibodies). Expression of the mRNAs of total cFN and the embryonic isoforms EIIIA and EIIIB was investigated, usingin situhybridization (ISH). The ratio between EIIIA containing fibronectin (EIIIA+–FN) mRNA and total cFN mRNA was determined using a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). cFN protein was present from day 4 until day 35 after infarction and was located around the area of infarction, in the epi- and endomyocardium and in the wall of larger vessels. pFN was found in the infarcted cardiomyocytes 1 day after the induction of the MI. From day 4 on pFN protein deposition was found in the border zone of the infarction and in the wall of larger vessels. pFN immunoreactivity remained present at high levels around the area of infarction and in the vessel wall throughout the entire period of investigation (90 days). From day 35 after the infarction pFN protein was detected in cardiomyocytes of the right ventricle and septum. cFN mRNA, determined byin situhybridization, was present in the border zone of the infarcted area as early as 1 day after MI, and its expression peaked at 4 days after MI. Four days after MI the mRNA's coding for both the embryonic isoforms EIIIA and EIIIB could also be detected in the same area. Because expression of the EIIIA isoform was more abundant than the EIIIB isoform we only determined the percentage of the EIIIIA containing isoform from total FN. EIIIA+mRNA was elevated 1 day after MI. We conclude that various fibronectin isoforms including the embryonic isoforms accumulate in the heart after MI. This suggests that these isoforms may play a role in the wound healing process in the left ventricle of the infarcted heart.

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The involvement of fibronectin in molecular and cellular interactions

Among the proteins of the animal organism, fibronectin (FN) occupies a special place due to the exceptional diversity and importance of biological properties. In 1948, Morrison et al. [69] for the first time it was discovered as part of the I fraction of blood plasma by Cohn, but interest in FN increased rapidly only in the 70s after its identification with one of the proteins of the outer cell membrane. At various times, FN has been described under numerous names that determined any property of this protein or its localization, for example, large external transformation-sensitive (LETS) protein, cold-insoluble globulin, cell adhesion factor, opsonic a2-SB-glycoprotein, anti-gelatin factor, etc. Currently, these names are almost not used, and the term "fibronectin" (fibra fiber, nectere bind) is adopted to refer to all forms of this protein cellular and extracellular, soluble and membrane-associated cells that form a population of immunologically related molecules with some differences in physicochemical and biological properties.