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PubMed1994

Fibronectin and lymphocytes in inflammatory tissue. Studies of blood and synovial fluid lymphocytes from patients with rheumatoid arthritis and other inflammatory arthritides.

Lymphocytes infiltrating tissues under chronic inflammatory conditions are often surrounded by deposits of fibronectin. We have studied the possibility that this reflects capacity of lymphocytes to synthesize fibronectin and compared lymphocytes from blood and synovial fluid with respect to fibronectin interactions. In vitro activated blood lymphocytes exhibited synthesis of a fibronectin-like molecule. Synovial fluid cells appeared to synthesize the same high molecular weight component spontaneously. Activated blood lymphocytes have cell surface fibronectin and surface components of lower molecular weight which could be immunoprecipitated with anti-fibronectin antibodies as well as by insolubilized collagen. Synovial fluid cells showed cell surface fibronectin as revealed by immunocytochemical detection but seemed to lack or have relatively small amounts of the low-molecular weight fibronectin-like surface components. Synovial fluid T cells from arthritis patients showed adhesion to fibronectin. Immunocytochemistry demonstrated presence of alpha 4 and alpha 5 beta 1 integrins at the surface of the synovial fluid T cells and RGD and LDV peptides inhibited adhesion of the cells to fibronectin. Noteworthy, a portion of synovial fluid cells with lymphocyte markers also bound to plastic. Blood lymphocytes from the same arthritis patients displayed relatively poor or negligible adhesion to fibronectin unless activated to blast transformation and did not attach to plastic. Taken together these results suggest that activated lymphocytes from blood and synovial fluid may use fibronectin of exogenous or endogenous origin when interacting with tissues during inflammatory processes. Furthermore, the presence at the lymphocyte surface of components of different molecular weight precipitated by anti-fibronectin antibodies suggests that fibronectin or its fragments can bind to the lymphocyte surface.

Arthritis↗
PubMed1994

Differential splicing of fibronectin pre-messenger ribonucleic acid during cardiac ontogeny and development of hypertrophy in the rat.

BACKGROUND: Fibronectin, an extracellular matrix protein, exists as multiple isoforms expressed in a time- and cell-dependent manner. Since the developmental pattern of fibronectin expression has not been determined in the heart, the first issue of this study was to investigate the expression of total fibronectin mRNA as well as its isoforms during cardiac ontogeny. In adults, pressure overload induces a shift towards the fetal form of proteins expressed by either muscle or nonmuscle cardiac cells. Fetal forms of fibronectin mRNA being found in smooth and nonmuscle cardiac cells soon after imposition of pressure overload, the pattern of fibronectin expression during the development of pathological growth was analyzed to determine whether the two conditions of cardiac growth resulted in an identical pattern of fibronectin expression. EXPERIMENTAL DESIGN: Total RNA were isolated from rat heart (a) during in utero and postnatal life and (b) at varying periods of time after imposition of a pressure overload induced by coarctation of the thoracic aorta in 25-day-old rats. Fibronectin-EIIIA+ or -EIIIB+ and total fibronectin mRNAs were quantitated by reverse transcription-polymerase chain reactions and dot-blot analysis, respectively. RESULTS: Fibronectin mRNA, abundant in the 14-day-old fetal heart, rapidly decreased during cardiac physiologic growth (> 5-fold); no changes in the fibronectin mRNA level was observed during the development of pressure-induced cardiac hypertrophy. The percentages of fibronectin transcripts containing EIIIA or EIIIB exons, very high in the early fetal heart (> 45%), harmoniously decreased during cardiac maturation (< 12%). Aortic coarctation resulted in an early, transient (12 to 48 hours) and preferential expression of fibronectin-EIIIA+ mRNA (approximately 40%). CONCLUSIONS: In rat heart, neither physiologic nor pressure-induced growth requires increased amounts of fibronectin mRNA but the growth conditions specifically modulated the fibronectin pre-mRNA splicing.

Animals↗
PubMed1996

Fibronectin in the tear film.

PURPOSE: Fibronectin plays an important role in corneal healing and has been detected previously in the tear film. To investigate the levels of fibronectin in normal human tears, the authors measured and compared fibronectin concentration in open-eye, closed-eye, and reflex tear fluid. The origin of fibronectin in the tear film was investigated by comparing fibronectin concentration in sequentially collected reflex tear samples with the concentrations of total protein and albumin in the same samples. METHODS: Open-eye and closed-eye tears were collected from 11 noncontact lens wearers. From 7 subjects, 20 uninterrupted reflex tear samples (10 microliters each) subsequently were collected, using the sneeze reflex method of stimulation, followed by an additional 10 nonstimulated tear samples (3 microliters each) immediately after cessation of stimulus. Enzyme-linked immunosorbent assays were used to determine fibronectin and albumin concentrations, and bicinchoninic acid protein assays were used to determine total protein concentration in each sample. RESULTS: Fibronectin concentration in open-eye tears (19 +/- 24 eta g/ml, range 3 to 78 eta g/ml) was significantly different (P = 0.004) from that in closed-eyes tears (4127 +/- 3222 eta g/ml, range 1177 to 11384 eta g/ml). In the first 50 microliters of reflex tears, fibronectin concentrations were low (10 +/- 23 eta g/ml), but they increased significantly (P = 0.028) after 100 microliters of reflex tears had been collected (220 +/- 126 eta g/ml). There was a further marked transient increase (767 +/- 946 eta g/ml) after cessation of stimulus. Total protein concentration in the same samples decreased significantly during reflex tear collection compared to open-eye tears, and it increased gradually after cessation of stimulus. Albumin concentration in the same samples, analyzed for two subjects only, showed a pattern similar to that for fibronectin. Dilation of conjunctival blood vessels was noted in all subjects after reflex tear collection. Administration of a topical vasoconstrictor in two subjects eliminated the increase in fibronectin concentration during reflex tearing but did not affect total protein concentration. Under reducing conditions, the molecular mass of fibronectin in open-eye and reflex tears was 240 kDa, identical to commercially available purified plasma fibronectin, whereas fibronectin in closed-eye tears was degraded into small molecular mass fragments. CONCLUSIONS: These findings suggest that fibronectin in tear fluid is derived from plasma and that the increase in concentration in closed-eye and reflex tear fluid is caused by leakage from dilated conjunctival blood vessels.

Adult↗
PubMed1978

Affinity of fibronectin to collagens of different genetic types and to fibrinogen.

Fibronectin, a fibroblast surface protein, was purified from human and chicken plasma and extracts of cultured chicken fibroblasts with affinity chromatography on gelatin coupled to Sepharose particles. A fibronectin-like protein was also isolated from the plasma of Torpedo fish. The collagen binding properties of fibronectin were studied with several genetically distinct collagens. Heat denatured types I, II, and III collagens were equal in their binding capacity and more active than the native collagens or A and B chains. Native type III collagen was more active than the other native collagens. Human and chicken fibronectins showed approximately the same pattern of specificity. Identical specificities were shown by the plasma and fibroblast forms of chicken fibronectin. Two cyanogen bromide peptides of the collagen alpha1 (II) chain, CB8 and CB12, derived from different parts of the chain, were active in fibronectin binding. A polymer of the tripeptide pro-gly-pro, and polyproline were inactive. Fibronectin also binds to fibrinogen and fibrin. Comparison of this binding to collagen binding showed that fibrinogen inhibited binding of fibronectin to collagen, but was less active than native collagen. Two other fibrous proteins, tropoelastin and keratin, did not bind fibronectin. The binding of fibronectin to fibrinogen was inhibited by collagen and incorporation of fibronectin into blood clot in the cold was inhibited by gelatin. These results suggest that the binding of fibronectin to collagen and fibrinogen depends on the same binding site in the fibronectin molecule. It is proposed that cell surface fibronectin mediates attachment of cells to the collagenous extracellular matrix and to a temporary fibrin matrix in a wound.

Animals↗
PubMed1982

Fibronectin binding to Staphylococcus aureus.

Bacteria are able to interact with a number of macromolecules which act as opsonins or tissue-adherence factors. Because soluble fibronectin may be important factors. Because soluble fibronectin may be important in the phagocytic removal of bacteria and insoluble fibronectin may serve as a bridge between bacteria and host tissues, we have characterized the binding of soluble plasma fibronectin to Staphylococcus aureus as a first step to understanding these interactions. The binding of 125I-fibronectin to clinical and laboratory strains of S. aureus was studied. Bound fibronectin was separated from free fibronectin by centrifugation. Specific binding was determined by subtracting the amount bound in the presence of excess fibronectin from the total amount bound. We found that (i) fibronectin bound saturably, irreversibly, and noncovalently to S. aureus when the binding reaction was carried out at pH 7.4 or greater; (ii) S. aureus harvested in logarithmic phase of growth from media buffered to pH 8.4, and from brain-heart infusion media which demonstrated the greatest number of fibronectin-binding sites; (iii) high molecular weight dextrans, fibrinogen, cyanogen bromide fragment 7 of collagen, cationic proteins, dibromide fragment 7 of collagen, cationic proteins, dithiothreitol, and protein A did not alt er fibronectin binding to S. aureus; (iv) nonsaturable binding occurred below pH 7.0 with peak binding occurring at pH 5.8; and (v) there were marked differences in the amounts of fibronectin that bind to different strains of S. aureus. S. aureus ATCC 25923, when harvested in logarithmic phase of growth from tryptic soy broth and tested for fibronectin binding in (2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline, pH 7.4, had 7500 binding sites/organism with an apparent association constant of 5.6 X 10(9) M-1.

Fibronectins↗
PubMed1986

Heparan sulphate proteoglycan as mediator of some adhesive responses and cytoskeletal reorganization of cells on fibronectin matrices: independent versus cooperative functions.

Fibronectin is a multifunctional glycoprotein which promotes the adhesion of a variety of cell types to extracellular matrices, including artificial tissue culture substrata. Biochemical analyses of substratum adhesion sites indicated important functions for cell-surface heparan sulphate proteoglycan (HS-PG) in directly mediating adhesive responses by the binding of heparan sulphate sequences to fibronectin. In addition, fibronectin has a binding domain for a cell surface 'receptor' (possibly a 140K glycoprotein) important in these responses. To differentiate the relative importance of these two binding activities, a proteolytically generated cell-binding fragment of fibronectin has been isolated which binds to the 140K 'receptor' but not to HS or to collagen. Platelet factor 4 (PF4), a tetravalent HS-binding protein, provides a model of the tetravalent HS-binding activity of fibronectin, as supported by affinity chromatography studies (these studies also reveal the complexity of HS-PG metabolism in adhesion sites). Responses are measured on substrata coated with the cell-binding fragment of fibronectin, intact fibronectin, or PF4, singly or in combination. Fibroblast-like BALB/c 3T3 cells form both close and tight-focal adhesive contacts with associated microfilament stress fibres on intact fibronectin. Whereas HS-PG binding appears to mediate the formation of close contacts and linear microfilament bundles, a cooperative relationship exists between the HS- and the cell-binding activities of the intact fibronectin molecule in the formation of focal contacts and stress fibres. Human dermal fibroblasts generate different adhesive responses on HS-binding or cell-binding substrata, which are dependent on whether cells have been grown in medium with ascorbate to maximize production of their own collagenous matrix. As with 3T3 cells, focal contact and stress fibre formations of dermal cells require both binding activities in the intact fibronectin molecule. A third system consists of neuroblastoma tumour cells which adhere and extend neurites on fibronectin. Cell-body adherence, but not neurite extension, occurs on HS-binding matrices whereas neurite extension requires only fibronectin's cell-binding activity; the responses of primary peripheral neurons were exactly the opposite and CNS neurons did not respond at all. These studies indicate the diversity of molecular mechanisms by which various cells interact with the multifunctional fibronectin molecule in order to perform specialized functions, as well as the independent or cooperative functions of heparan sulphate proteoglycan on the cell surface in mediating these responses.

Animals↗
PubMed1984

Characterization of synovial fluid fibronectin from patients with rheumatic inflammatory diseases and healthy subjects.

Synovial fluid fibronectin from normal subjects and from patients who have rheumatic inflammatory diseases has been studied and compared with plasma fibronectin. The average fibronectin concentration in synovial fluids from normal subjects was 172 +/- 69 micrograms/ml; it was 721 +/- 315 and 556 +/- 349 micrograms/ml in synovial fluids from patients with rheumatoid arthritis and osteoarthritis, respectively. This is the first report on fibronectin concentrations in normal synovial fluids. Synovial fluid fibronectin from healthy subjects and patients with rheumatoid arthritis or osteoarthritis showed a molecular weight identical to that of plasma fibronectin. All normal and pathologic synovial fluid fibronectins showed a remarkably lower electrophoretic mobility compared with that of plasma fibronectin, when separated according to net molecular charge on agarose gel. Peptides from thermolysin digests of fibronectin from plasma and synovial fluid, when compared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed distinct differences. These data demonstrate that synovial fluid fibronectin represents a molecular form which is structurally different from that of plasma fibronectin. This suggests that synovial fluid fibronectin is locally synthesized, possibly by a cell type which differs from that responsible for the production of the plasmatic fibronectin pool.

Aged↗
PubMed2006

Identification of fibronectin neoepitopes present in human osteoarthritic cartilage.

OBJECTIVE: Fibronectin fragments are present at high concentrations in the cartilage of patients with rheumatoid arthritis and patients with osteoarthritis (OA) and have been shown to promote cartilage catabolism in human cartilage cultures, suggesting that fibronectin fragments participate in the initiation and progression of arthritic disease. This study was undertaken to 1) identify the major fibronectin fragments in human OA cartilage and confirm their ability to elicit cartilage catabolism, 2) identify the cleavage sites in fibronectin and generate the corresponding neoepitope antibodies, and 3) explore the utility of fibronectin neoepitopes as biomarkers. METHODS: Fibronectin fragments were purified from human OA cartilage using affinity chromatography; their N-termini were then identified by sequencing. Bovine nasal cartilage was treated with affinity-purified fibronectin fragments and assayed for aggrecan breakdown by monitoring the release of glycosaminoglycans and the aggrecan neoepitope 1771AGEG. Fibronectin neoepitopes were detected by Western blotting in cytokine-treated media of human cartilage explants, and by immunohistochemical analyses of human OA cartilage. RESULTS: Multiple fibronectin fragments were isolated from human OA cartilage, and all contained the N-terminus 272VYQP. These fragments induced aggrecanase-mediated cartilage catabolism in bovine cartilage explants. Fibronectin fragments with the N-terminus 272VYQP and fragments with the C-terminus VRAA271 were detected following cytokine treatment of human cartilage extracts. These neoepitopes localized with areas of aggrecan loss in OA cartilage. CONCLUSION: Human OA cartilage contains fibronectin fragments with catabolic activity and a major cleavage site within fibronectin. This study is the first to characterize fibronectin neoepitopes in OA cartilage, suggesting that they may represent a novel biomarker of arthritis.

Aggrecans↗
PubMedDate not supplied

Sinusoidal endothelial cells from guinea pig liver synthesize and secrete cellular fibronectin in vitro.

Endothelial liver cells were obtained from guinea pig by enzymatic digestion and centrifugal elutriation. Cells were cultured on gelatin and fibronectin pretreated culture vessels. Endothelial cells were characterized by phase-contrast microscopy, electron microscopy and the presence of Factor VIII-related antigen. Fibronectin secretion was determined in cell-free supernatants by a sensitive and specific ELISA and localized on fixed cultured cells by immunofluorescence. [35S]Methionine endogeneously labeled fibronectin was immunoprecipitated from supernatants and cellular lysates and displayed on sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. After attachment to the culture vessel, one day after plating, endothelial cells start to produce fibronectin as measured by ELISA and demonstrated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Secretion of fibronectin increases as cells proliferate to form a confluent monolayer. By immunofluorescence, fibronectin is visualized inside permeabilized cells and as a fibrillar network on the cell surface. Underneath the cell bodies, fibronectin-positive material is present as short strands. From supernatants and cellular lysates, fibronectin is immunoprecipitated with an apparent Mr of about 235,000 obviously larger than plasma fibronectin with an Mr of 220,000, which behaves electrophoretically like fibronectin isolated from early hepatocyte cultures. As endothelial cells incorporate [3H]fucose in fibronectin, whereas hepatocytes do not, we conclude that endothelial cells in contrast to hepatocytes produce cellular fibronectin. Endothelial cells, therefore, are probably the cellular source of the fibronectin present in the space of Disse. The significance of this finding with respect to fibrotic liver disease is discussed.

Animals↗
PubMed1986

Substratum influence on collagen and fibronectin biosynthesis by arterial smooth muscle cells in vitro.

Collagen, fibronectin, and nonfibrous protein biosynthesis were examined in cultures of rabbit arterial smooth muscle cells grown on tissue culture plastic precoated either with rabbit plasma fibronectin or bovine serum albumin. Cells seeded into fibronectin-coated wells appeared to reach confluence more quickly than counterparts grown on albumin-coated surfaces. Measurement 3H-thymidine incorporation into DNA by these cultures suggested that this was probably a consequence of more rapid and efficient cell attachment rather than an increased rate of proliferation of smooth muscle cells grown on fibronectin. In preconfluent cultures, the rates of collagen and fibronectin biosynthesis were reduced to 34 and 57%, respectively, on a per-cell basis in cultures grown on fibronectin-coated surfaces compared with cells grown on albumin-coated plasticware. In preconfluent cultures grown on fibronectin-coated surfaces, a greater percentage of the total fibronectin synthesized was incorporated into the cell layer. The distribution of newly synthesized collagen between culture medium and cell layer, however, was not affected by alteration of substratum composition. There was no difference in the rate of synthesis of noncollagen proteins between the two groups of preconfluent cells. In postconfluent cultures the rates of collagen and fibronectin biosynthesis were equivalent in both albumin- and fibronectin-treated cultureware. In preconfluent cultures, analyses of procollagens showed that the overall amounts of both types I and III procollagens were reduced in fibronectin-treated wells, indicating the reduction in collagen synthesis to be general and not type-specific. Although type V procollagen biosynthesis was not detected in either preconfluent group, it was found in postconfluent cultures. The reduction of fibronectin synthesis in cells grown in fibronectin-coated wells was significant as early as 4 hours after plating. Together, these findings suggest that cultured arterial smooth muscle cells are capable of deriving information from their substratum and regulating the biosynthetic rates of extracellular matrix components in response to the immediate needs of the cell.

Animals↗
PubMed1986

Incorporation of purified plasma fibronectin into explants of articular cartilage from disease-free and osteoarthritic canine joints.

The purpose of this study was to determine if articular cartilage was able to accumulate fibronectin, a large molecule of 440,000 daltons, from the external medium, and if so, to compare the extent of accumulation by normal and osteoarthritic cartilage and to localize the sites of fibronectin accumulation within the articular cartilage. The uptake of canine serum albumin, another protein present in plasma and synovial fluid with a lower molecular weight (67,000 daltons) and a lower pI, was compared. Purified plasma fibronectin and canine albumin were labelled with 125I or N-hydroxysuccinimidobiotin by standard procedures and incubated with articular cartilage explants. The 125I-fibronectin that had bound to cartilage components was extracted with 4 M urea, and both extract and cartilage residues were counted. Cartilage accumulated fibronectin to a greater extent than albumin. For normal cartilage, a level of saturation appeared to be reached at an external concentration for fibronectin of about 150 micrograms/ml. Degenerated cartilage accumulated about 10-fold more fibronectin than normal cartilage. Biotinylated fibronectin was localized within frozen sections of articular cartilage by probing with peroxidase-linked avidin. Fibronectin accumulation in normal cartilage was restricted to the articular surface and the cut edge. In degenerated cartilage, penetration of fibronectin was more extensive but proceeded only from the articular surface. Staining of adjacent sections with peroxidase-linked antifibronectin antibody confirmed previous observations that endogenous fibronectin is present throughout the cartilage matrix. The possibility that synovial fluid fibronectin could be a source of cartilage fibronectin, especially in degenerated cartilage, was discussed.

Animals↗
PubMed1998

Effects of fibronectin on articular cartilage chondrocyte proteoglycan synthesis and response to insulin-like growth factor-I.

Fibronectin, a ubiquitous glycoprotein of the extracellular matrix, serves as a substrate for cell attachment. Binding to fibronectin through cell-surface receptors promotes a flattened cell shape, stimulates the phosphorylation of intracellular protein, and changes the pattern of gene expression. Although fibronectin is abundant in normal articular cartilage, its effects on chondrocytes are not well understood. Proteolytic fragments of fibronectin stimulate the catabolism of matrix in articular cartilage and may promote the degeneration of cartilage in osteoarthritis; however, intact fibronectin may regulate other aspects of matrix metabolism, including matrix synthesis. To determine whether intact fibronectin affects the synthetic activity of chondrocytes, as well as to determine the responses of chondrocytes to the anabolic growth factor insulin-like growth factor-I, we compared the incorporation of [35S] by articular chondrocytes of the rat cultured in the presence and absence of commercially prepared cellular fibronectin and 0, 10, or 100 ng/ml recombinant human insulin-like growth factor-I. Monolayer and alginate suspension cultures were compared to determine whether responses differed under conditions in which fibronectin promoted a flattened cell shape (monolayer culture) and under those in which cells maintained a spherical shape (alginate culture). In alginate cultures, fibronectin alone stimulated the incorporation of [35S]. Fibronectin with 10 ng/ml insulin-like growth factor-I had additive effects in alginate culture, producing the maximum incorporation of [35S]. In monolayer cultures, fibronectin did not stimulate incorporation and blocked stimulation by 100 ng/ml insulin-like growth factor-I. The cells from the monolayer culture were much less active synthetically (at all doses of the growth factor) than those cultured in alginate. Thus, fibronectin enhanced proteoglycan synthesis and the response to insulin-like growth factor-I in alginate but inhibited the response to the growth factor in monolayers. These observations suggest intact fibronectin may contribute to the maintenance or repair of the matrix of articular cartilage by stimulating proteoglycan synthesis.

Alginates↗
PubMed1995

Different roles for fibronectin in the generation of fore and hind limb precartilage condensations.

Fibronectin expression and spatiotemporal distribution were examined in relation to the distinctive patterns of mesenchymal condensation and chondrogenesis seen in high-density serum-free cultures of chicken wing and leg bud precartilage cells. More fibronectin protein was produced on a per cell basis by leg than by wing mesenchyme, both in freshly isolated tissue and during the prechondrogenic condensation period in culture, where the difference was twofold. The quantitative difference in fibronectin expression in freshly isolated wing and leg mesenchyme was also seen at the level of total and poly (A)+ RNA. During the condensation phase, fibronectin was distributed in the wing and leg mesenchymal cultures in a way that prefigured the eventual distribution of cartilage in these cultures: in wing cultures condensations were broad and flat, and rich in diffusely organized fibronectin; in leg cultures, condensations were compact and spheroidal, and contained abundant deposits of fibronectin. In addition, the leg condensations were connected by long fibronectin-rich fibers. Transient treatment with TGF-beta early during the culture period led to increase in fibronectin production and expansion of condensations in both wing and leg cultures. Leg mesenchyme was more responsive to transforming growth factor-beta than wing mesenchyme with respect to fibronectin production, and this was reflected in a greater enhancement of cartilage formation in later cultures. Treatment of cultures with monoclonal antibody 304 directed against the amino-terminal heparin-binding domain of fibronectin inhibited condensation formation and reduced chondrogenesis in wing mesenchyme, but left these two processes unchanged in leg mesenchyme, despite disruption by the antibody of the leg-specific fibronectin fibers. These studies indicate that for both wing and leg mesenchyme the morphology, extent, and spatiotemporal regulation of precartilage condensation and subsequent chondrogenesis closely parallels the deposition of fibronectin. But whereas the interaction between cells and fibronectin in wing bud mesenchyme is mediated in part by the protein's amino-terminal domain, this domain does not appear to be involved in analogous interactions in leg bud mesenchyme.

Animals↗
PubMed1992

Local production of fibronectin by ectopic human retinal cells.

The distribution of fibronectin mRNA and fibronectin in adult human retina and epiretinal membranes was investigated by in situ hybridisation and immunohistochemical techniques. The cells in normal adult retina contained little or no fibronectin mRNA and the retina only showed fibronectin immunoreactivity in retinal vessels. The cells in detached neuroretina did not contain fibronectin message but the vitreoretinal interface of the detached retina exhibited variable fibronectin immunoreactivity. Retinal glia, retinal pigment epithelium and fibroblast-like cells in membranes at the vitreoretinal juncture (epiretinal membranes) showed variable labelling with the fibronectin mRNA probe and all the membranes immunostained for fibronectin. No difference could be detected between membrane cell types in the intensity of labelling with the mRNA probe or for fibronectin immunoreactivity. The results indicate that cells in situ in attached and detached adult human retina do not produce fibronectin. Although fibronectin at the vitreoretinal juncture in retinal detachment is probably partly derived from plasma fibronectin resulting from breakdown of the blood-retinal barrier, ectopic retinal cells produce fibronectin and contribute to the glycoprotein epiretinal membranes.

Adolescent↗
PubMed1992

Rat hepatocytes in primary culture synthesize and secrete cellular fibronectin.

Fibronectins, involved in cell-matrix interactions and cell attachment, are glycoproteins which show a remarkable heterogeneity, due to alternative splicing. The type III-related domains, ED-A and ED-B, are present in cellular fibronectin in a variety of ratios whereas they are absent in circulating plasma fibronectin. Fibronectin synthesis by hepatocytes which are accepted as suppliers of plasma fibronectin was studied in primary cultures during a 6-day culture period. Using site-specific antibodies we demonstrate that rat hepatocytes are also able to synthesize and secrete fibronectin bearing the ED-A domain from Day 3 on after inoculation. By immunocytological characterization of the hepatocyte monolayer with antibodies directed against desmin, laminin, collagen IV, alpha-SM-actin, or ED-1 or factor VIII-related antigen, contaminating mesenchymal hepatic cell-types as a source for cellular fibronectin production could be ruled out. Dexamethasone treatment caused enhanced fibronectin synthesis and cellular fibronectin was already detectable at Day 1 after plating. Elevation of cellular fibronectin synthesis after prolonged culture-terms and by dexamethasone could also be demonstrated on mRNA steady-state level, using ED-A cDNA as a probe in hybridization analysis. Dot blot hybridisation proved a prominent response of cellular fibronectin mRNA level to dexamethasone at Day 1 when dexamethasone treatment resulted in an increased contribution of ED-A-positive fibronectin transcripts to total fibronectin mRNA level.

Animals↗
PubMed1993

V(+)-fibronectin expression and localization prior to gastrulation in Xenopus laevis embryos.

The V-region represents one of three alternatively spliced segments in Xenopus fibronectin. Here, we identify this V-region as binding epitope of the monoclonal antibody (MAb 6D9) that we generated against Xenopus plasma fibronectin. By the use of this antibody we obtained new results that change the present view of the fibronectin expression pattern before gastrulation: (1) the V(+)-fibronectin is the major isoform expressed during early development since only a single fibronectin band is found in Western blots up to tadpole stages. (2) In contrast to previously published data we demonstrate that fibronectin expression is induced by progesterone during oocyte maturation. (3) During cleavage stages the protein is stored in the cytoplasm where it is predominantly associated with plasma membranes. Immunoelectronmicroscopy reveals that V(+)-fibronectin is present at the surface of animal pole blastomeres and secreted into intercellular spaces. This extracellular localization of fibronectin is predominantly observed in the marginal zone, surrounding single cells of the outer cell layer baso-laterally. In the vegetal hemisphere V(+)-fibronectin is restricted to the cytoplasm and accumulated at plasma membranes. With the onset of gastrulation the intracellular and membrane associated fibronectin disappears and fibronectin becomes detectable at the blastocoel roof. Since reaggregation of dissociated blastula cells was not blocked by addition of GRGDS peptide or antibodies against fibronectin, we assume that the early expression and secretion of fibronectin serves as store to allow a rapid matrix assembly with onset of mesodermal cell migration.

Alternative Splicing↗
PubMed1995

Conformational transitions in the cell binding domain of fibronectin.

Plasma fibronectin readily changes shape in response to environmental conditions which may, in turn, lead to differential expression of its multiple functional sites. To test this possibility, the expression of two of the type III modules within cell binding domain of fibronectin was assessed with monoclonal antibodies (mAb). Utilizing proteolytic and recombinant fragments of plasma fibronectin, the epitopes recognized by mAbIII-9 and mAbIII-10 were localized to the ninth and tenth (RGD-containing) type III repeats of fibronectin, respectively. Both mAb inhibited the adhesion of platelets to immobilized fibronectin, suggesting that the recognized epitopes resided in close spatial proximity to the cell binding sites. Radioimmunoassay and Scatchard analyses showed that, in solution, each dimeric fibronectin molecule bound two mAbIII-9 but only one mAbIII-10 molecule (ionic strength 0.15, pH 7.4). The binding of a single mAbIII-10 per fibronectin molecule was verified by electron microscopy. Heparin, heparan sulfate, gangliosides (but not chondroitin sulfates A and B and hyaluronic acid), and self-association increased the apparent affinity of mAbIII-10 for soluble fibronectin. Adsorption of fibronectin onto a polystyrene surface resulted in the appearance of an additional binding site for mAbIII-10. MAbIII-9 binding also was altered by fibronectin immobilization. These results suggest that the deposition of fibronectin and its interaction with components of the extracellular matrix can modulate the expression of the cell binding domains including the RGDS-containing type III repeat. Exposure of the second tenth type III repeat within the fibronectin dimer, as a result of unfolding on a surface, could contribute to the enhanced adhesiveness of adsorbed fibronectin.

Antibodies, Monoclonal↗
PubMed1981

Comparative studies on amniotic fluid and plasma fibronectins.

Human fibronectin was isolated from second-trimester amniotic fluid, from amniotic fluid obtained at term and from adult plasma. The amniotic-fluid fibronectins had a slightly higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than the plasma fibronectin. Early- and late-amniotic-fluid fibronectin had 9.5 and 9.6% carbohydrate respectively, whereas plasma fibronectin had 5.8%. The amniotic-fluid fibronectins had similar mannose and sialic acid contents to plasma fibronectin, but greater amounts of glucosamine, galactosamine, galactose and fucose. There were no detectable differences in the amino-acid composition of amniotic-fluid and plasma fibronectins, and the patterns of peptides obtained after tryptic digestion of fibronectin from the two sources showed extensive similarities. Fibronectins from plasma and amniotic fluid were equally active in promoting cell attachment and were immunologically indistinguishable. These results show that fibronectin from amniotic fluid is more heavily glycosylated than plasma fibronectin or previously analysed fibronectins from cultured fibroblasts. The observed differences in glycosylation may be related to cell type and/or stage of development.

Amino Acids↗