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PubMed1995

[Reliability of the detection of fetal fibronectin using monoclonal antibody FDC-6].

Detection of amniotic fluid in the vagina is commonly used to diagnosed rupture of amniotic membranes. In 1991 an immunoassay was introduced which is based on the detection of a fetal fibronectin isoform using the monoclonal antibody FDC-6. Fetal fibronectin is said to be absent from normal adult tissues and plasma, but present in fetal tissues. The low concentration of fetal fibronectin in maternal plasma is said not to interfere with the test results. Theoretically, demonstration of significant amounts of fetal fibronectin in vaginal secretions may be considered an unequivocal sign of leakage of amniotic fluid or, at least, of pre-rupture stretching of the membranes. We evaluated results of the membrane immunoassay and enzyme immunoassay, which are both based on the monoclonal antibody FDC-6. Fetal fibronectin has been determined in a clinically unambiguous context in amniotic fluid as well as in vaginal swabs and blood samples. In amniotic fluid, all membrane immunoassays performed were positive. The concentration of fetal fibronectin during the third trimester of pregnancy was lower than in the second trimester. Only 9/15 cases were positive for fetal fibronectin if vaginal swabs were taken 12 hours after unequivocal rupture of membranes and no amniotic fluid was visible in the vagina. Vaginal swabs of women with regular contractions showing cervical dilation of > 2 cm were positive for fetal fibronectin in all cases (20/20) whereas no fetal fibronectin could be determined in vaginal swabs obtained from clinically inapparent pregnant women. In all blood samples of pregnant women fetal fibronectin was detected and, surprisingly, also in blood samples of non-pregnant women, as well as in vaginal swabs of non-pregnant women with vaginal bleeding. In cases of unequivocal rupture or intactness of membranes, the results of both assays correspond well with the clinical findings. 12 hours after rupture of membranes the test may become negative if no amniotic fluid is visible in the vagina. As all blood samples had been positive for fetal fibronectin we conclude that vaginal bleeding will probably interfere with the test. The most surprising result is the apparent presence of fetal fibronectin in the blood samples of non-pregnant women. This observation can only be explained by a cross-reaction of the FDC-6 antibody with hepatic fibronectin.

Adult↗
PubMed1981

Deposition of plasma fibronectin in tissues.

Tissue distribution of human fibronectin injected intravenously into mice was studied by using immunofluorescence with species-specific antisera to human and mouse fibronectins. Human fibronectin was detected in the tissues of mice injected with human fibronectin. The distribution of the injected fibronectin was indistinguishable from that of mouse fibronectin. The staining for human fibronectin in the livers of injected mice was unaffected by perfusion of the livers prior to the preparation of tissue sections, and human IgG injected in mice as a control was not detectable in tissues by immunofluorescence. Mice were also injected with fragments of fibronectin with molecular weights close to 200,000. These fragments lack parts of the NH2 and COOH termini of the fibronectin polypeptide, including the part with the interchain disulfide bond(s), but retain the ability to bind to collagen and heparin and the ability to mediate cell attachment. They showed essentially no tissue incorporation, suggesting that the integrity of the fibronectin molecule is important for the accumulation of fibronectin in tissues. The incorporation of injected fibronectin into tissues demonstrated here suggests that circulating fibronectin contributes to the extracellular matrix of tissues.

Animals↗
PubMed1999

Identification of protein-disulfide isomerase activity in fibronectin.

Assembly and degradation of fibronectin-containing extracellular matrices are dynamic processes that are up-regulated during wound healing, embryogenesis, and metastasis. Although several of the early steps leading to fibronectin deposition have been identified, the mechanisms leading to the accumulation of fibronectin in disulfide-stabilized multimers are largely unknown. Disulfide-stabilized fibronectin multimers are thought to arise through intra- or intermolecular disulfide exchange. Several proteins involved in disulfide exchange reactions contain the sequence Cys-X-X-Cys in their active sites, including thioredoxin and protein-disulfide isomerase. The twelfth type I module of fibronectin (I12) contains a Cys-X-X-Cys motif, suggesting that fibronectin may have the intrinsic ability to catalyze disulfide bond rearrangement. Using an established protein refolding assay, we demonstrate here that fibronectin has protein-disulfide isomerase activity and that this activity is localized to the carboxyl-terminal type I module I12. I12 was as active on an equal molar basis as intact fibronectin, indicating that most of the protein-disulfide isomerase activity of fibronectin is localized to I12. Moreover, the protein-disulfide isomerase activity of fibronectin appears to be partially cryptic since limited proteolysis of I10-I12 increased its isomerase activity and dramatically enhanced the rate of RNase refolding. This is the first demonstration that fibronectin contains protein-disulfide isomerase activity and suggests that cross-linking of fibronectin in the extracellular matrix may be catalyzed by a disulfide isomerase activity contained within the fibronectin molecule.

Base Sequence↗
PubMed1981

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells.

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.

Animals↗
PubMed1986

Factor XIII as a modulator of plasma fibronectin alterations during experimental bacteremia.

Fibronectin is found in plasma as well as in association with connective tissue and cell surfaces. Depletion of plasma fibronectin is often observed in septic trauma and burned patients, while experimental rats often manifest hyperfibronectinemia with sepsis. Since Factor XIII may influence the rate of clearance and deposition of plasma fibronectin into tissues, we evaluated the temporal changes in plasma fibronectin and plasma Factor XIII following bacteremia and RE blockade in rats in an attempt to understand the mechanism leading to elevation of fibronectin levels in bacteremic rats, which is distinct from that observed with RE blockade. Clearance of exogenously administered fibronectin after bacteremia was also determined. Rats received either saline, Pseudomonas aeruginosa (1 X 10(9) organisms), gelatinized RE test lipid emulsion (50 mg/100 gm B.W.), or emulsion followed by Pseudomonas. Plasma fibronectin and Factor XIII were determined at 0, 2, 24, and 48 hours post-blockade or bacteremia. At 24 and 48 hr following bacteremia alone or bacteremia after RE blockade, there was a significant elevation (p less than 0.05) of plasma fibronectin and a concomitant decrease (p less than 0.05) of plasma factor XIII activity. Extractable tissue fibronectin from liver and spleen was also increased at 24 and 48 hours following R.E. blockade plus bacteremia. In addition, the plasma clearance of human fibronectin was significantly prolonged (p less than 0.05) following bacterial challenge. Infusion of activated Factor XIII (20 units/rat) during a period of hyperfibronectinemia (908.0 +/- 55.1 micrograms/ml) resulted in a significant (p less than 0.05) decrease in plasma fibronectin (548.5 +/- 49.9 micrograms/ml) within 30 min. Thus Factor XIII deficiency in rats with bacteremia may contribute to the elevation in plasma fibronectin by altering kinetics associated with the clearance of fibronectin from the blood.

Animals↗
PubMed2000

Fibronectin and its fragments increase with degeneration in the human intervertebral disc.

STUDY DESIGN: This laboratory-based experiment correlates fibronectin content of intervertebral disc with a morphologic grade of degeneration. OBJECTIVES: To correlate the fibronectin content of the anulus fibrosus and nucleus pulposus with a gross morphologic grade of disc degeneration, and to determine the molecular size of the extractable fibronectin. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration increases with age and can lead to low back pain. Fibronectin helps to organize the extracellular matrix and provides environmental cues by interaction with cell surface integrins. In other tissues, its synthesis is elevated in response to injury. Fibronectin fragments can stimulate cells to produce metalloproteases and cytokines and inhibit matrix synthesis. METHODS: In this study, 17 anuli fibrosis and 18 nuclei pulposus from 11 spines were graded by Thompson's gross morphologic scale. Fibronectin was sequentially extracted with 4 mol/L guanidine hydrochloride and trypsin, and then quantitated by enzyme-linked immunoassay. The size of extractable fibronectin was determined by Western blot analyses. RESULTS: The fibronectin content of the disc increased with grade and was significantly elevated between Grades 3 and 4. The percentage of extractable fibronectin varied widely, but it was more extractable from the nucleus. In both the nucleus and anulus, 30% to 40% of the extractable fibronectin existed as fragments. Many of the fragments contained functional heparin or collagen-binding sites. CONCLUSIONS: Fibronectin is elevated in degenerated discs and frequently present as fragments. Elevated levels of fibronectin suggest that disc cells are responding to the altered environment. Fibronectin fragments resulting from normal or enhanced proteolytic activity could be a mechanism that induces the cell to degrade the matrix further.

Adolescent↗
PubMed1993

Role of fibronectin in staphylococcal colonisation of fibrin thrombi and plastic surfaces.

The adhesive glycoprotein fibronectin has been proposed as a mediator of adherence of certain gram-positive cocci to host cells and fibrin thrombi. This study compared the role of soluble and immobilised fibronectin in the adherence of coagulase-negative staphylococci (CNS) and Staphylococcus aureus to fibrin thrombi and plastic surfaces. Adherence of S. epidermidis to fibrin thrombi was significantly reduced when fibronectin was removed from the plasma used for thrombus preparation. Adherence was restored through restitution of fibronectin. S. epidermidis also adhered substantially more to plastic surface coated with fibronectin than to non-coated plastic. Increased adherence of CNS to plastic was also observed after coating with the 29-kDa N-terminal fragment of fibronectin. Soluble fibronectin did not affect the adherence of CNS to fibrin thrombi or plastic surfaces. The adherence of S. aureus to fibrin thrombi was significantly increased by the addition of soluble fibronectin, but not by incorporation of fibronectin into the clot. These results indicate that the binding of fibronectin is an important factor in the adherence of staphylococci to fibrin clots and plastic surfaces and, thus, colonisation of these surfaces. However, the two species of staphylococci seem to employ different mechanisms of fibronectin-mediated adherence: S. epidermidis interacts mainly with fibronectin incorporated in fibrin clots or immobilised on implanted synthetic materials, whereas S. aureus adheres to the fibrin matrix through binding of soluble fibronectin present in wound exudates.

Albumins↗
PubMed1991

Human keratinocytes adhere to two distinct heparin-binding synthetic peptides derived from fibronectin.

Fibronectin is present at the dermal-epidermal junction in normal skin and is increased in skin tissues in inflammatory diseases, skin cancers, and wound repair. The present studies focused on further characterizing the interaction between fibronectin and keratinocytes, specifically addressing whether human keratinocytes utilize multiple adhesion promoting sequences within fibronectin. Initially, direct cell-binding assays were utilized in which keratinocyte adhesion to plastic substrata coated with fibronectin or proteolytic fragments of fibronectin was quantified. Intact fibronectin, a 75-kD proteolytic fragment containing the RGD sequence, and 33/66-kD cell adhesion/heparin binding fragments lacking the RGD sequence derived from the A and B chains of fibronectin, all promoted keratinocyte adhesion in a concentration-dependent manner. To further define putative cell-binding domains within the 33/66-kD fibronectin fragments, we studied three chemically synthesized peptides derived from the amino acid sequence of the 33-kD fragment of the fibronectin A chain: FN-C/H-I (YEKPGSPPREVVPRPRPGV), FN-C/H-II (KNNQKSEPLIGRKKT), and CS1 (DELPQLVTLPHPNLHGPEILDVPST). Substrata coated with either FN-C/H-I or FN-C/H-II promoted keratinocyte adhesion in a concentration-dependent and saturable manner, whereas peptide CS1 promoted no significant keratinocyte adhesion. In solution, both exogenous FN-C/H-I and FN-C/H-II partially inhibited keratinocyte adhesion to the 33/66-kD fibronectin fragments. Furthermore, antibodies prepared against these peptides also inhibited keratinocyte adhesion to the 33/66-kD fibronectin fragments. These data indicate that keratinocyte adhesion to fibronectin is mediated by multiple distinct amino acid sequences, at least two of which are localized to the carboxy-terminal heparin binding domain of fibronectin.

Antibodies↗
PubMed1981

Fibronectin.

The current knowledge of the structure, expression and functions and fibronectin is reviewed. Fibronectin is a high molecular glycoprotein present in the blood, connective tissue and at cell surface. It is synthesized by many types of differentiated cells and is believed to be involved in the attachment of cells to the surrounding extracellular matrix. Fibronectin has affinity to the other main components of extracellular matrix, collagen and glycosaminoglycans. It also interacts with cell surfaces as shown by the fact that fibronectin-collagen complexes, or fibronectin alone when insolubilized on a surface such as plastic, enhances the attachment of various types of cells to such surfaces. It seems that fibronectin, through its binding to collagen and to the cell surface, forms a bridge between the cell and its surrounding matrix. Circulating fibronectin may participate in the formation of extracellular matrix in tissues. It may also function as a nonspecific opsonin design to facilitate the uptake of tissue debris by phagocytic cells. Studies aimed at elucidating the significance of fibronectin in physiological phenomena and in disease have only just begun. The abundance of fibronectin in basement membrane structures and the development changes observed in its expression lead one to believe that the attachment (or lack of it) of cells to fibronectin plays a significant role in morphogenetic events or in normal development. Malignantly transformed cells tend to lack cell-associated fibronectin. The significance of the lack of surface fibronectin in transformed cells is a matter of some controversy, but it fibronectin is indeed the main mechanism that anchors cells to the extracellular matrix, disturbances of this mechanism could play an important role in malignancy and many other diseases.

Cells↗
PubMed1986

Binding of Streptococcus pyogenes to soluble and insoluble fibronectin.

The interaction of soluble and insoluble fibronectin with Streptococcus pyogenes was investigated. Soluble fibronectin bound to S. pyogenes in a dose-dependent and irreversible manner. Lipoteichoic acid competitively inhibited the binding of fibronectin to S. pyogenes but had little effect on the binding of fibronectin to staphylococci or pneumococci. The phase of growth of the streptococci had a slight effect on binding of fibronectin, with optimal binding occurring in the late log phase. S. pyogenes cells bound to fibronectin immobilized on microtiter plates in a dose-dependent and saturable manner. Both soluble fibronectin and lipoteichoic acid inhibited the binding of streptococci to immobilized fibronectin, suggesting that streptococci interact with soluble and insoluble fibronectin in a similar manner. Antibodies to fibronectin blocked the attachment of streptococci to immobilized fibronectin, whereas normal serum had no effect. Adherence of streptococci to buccal epithelial cells was inhibited by antibodies to fibronectin, but not by normal sera or by antibodies to buccal epithelial cells. The data suggest that lipoteichoic acid on the surface of S. pyogenes binds to fibronectin exposed on the host cell and that such binding mediates the attachment of streptococci to host cells.

Adhesiveness↗
PubMed1983

Binding of streptococcal lipoteichoic acid to fatty acid-binding sites on human plasma fibronectin.

The ability of Streptococcus pyogenes lipoteichoic acid and palmitic acid to bind to purified human plasma fibronectin was investigated. Initial studies indicated that intact fibronectin formed soluble complexes with lipoteichoic acid, resulting in a change in the mobility of fibronectin in an electrical field. Fibronectin covalently linked to agarose beads bound radiolabeled lipoteichoic acid in the acylated form but not in the deacylated form. An 18-M excess of fibronectin inhibited binding of lipoteichoic acid to the immobilized protein by 92%. Fibronectin-bound [(3)H]lipoteichoic acid could be specifically eluted with unlabeled lipoteichoic acid, as well as by fatty acid-free serum albumin. Serum albumin, which is known to contain fatty acid-binding sites capable of binding to the lipid moieties of lipoteichoic acid, inhibited the binding of lipoteichoic acid to fibronectin in a competitive fashion. The fibronectin-bound lipoteichoic acid could be eluted by 50% ethanol and various detergents but not by 1.0 M NaCl, various amino acids, or sugars. Similarly, radiolabeled palmitic acid adsorbed to fibronectin could be eluted with 50% ethanol but not with 1.0 M NaCl. Fibronectin adsorbed to a column of palmityl-Sepharose was eluted with 50% ethanol in 0.5% sodium dodecyl sulfate but not with 1.0 M NaCl or 1% sodium dodecyl sulfate alone. The binding of lipoteichoic acid to fibronectin followed first-order kinetics and was saturable. A Scatchard plot analysis of the binding data indicated a heterogeneity of lipoteichoic acid-binding sites similar to that previously found for serum albumin. Nevertheless, fibronectin contains at least one population of high-affinity binding sites for lipoteichoic acid. The binding affinity (nKa approximately 250 muM(-1)) is 2 orders of magnitude greater than the binding affinity of serum albumin. These data suggest that human plasma fibronectin contains specific binding sites for fatty acids and that lipoteichoic acid binds to these sites by way of its glycolipid moiety.

Binding Sites↗
PubMed1988

Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.

Cycloheximide↗
PubMed1984

Fibronectin deposition in delayed-type hypersensitivity. Reactions of normals and a patient with afibrinogenemia.

During development of delayed hypersensitivity (DH) skin reactions, fibronectin accumulates in two distinct sites: (a) the dermal interstitium in a pattern similar to fibrin and with a time course similar to that of fibrin deposition and mononuclear cell infiltration, and (b) blood vessel walls in a pattern suggestive of basement membrane staining and with a time course similar to that of endothelial cell proliferation. In vitro fibronectin can bind to monocytes or endothelial cells and simultaneously bind to fibrin or collagen matrices; by such interaction in vivo it may affect cell migration or proliferation. Thus, fibronectin deposition in DH reactions may facilitate cell-matrix interactions; however, the possibility exists that extravascular fibronectin accumulation may be only secondary to interstitial fibrin clot formation, and that blood vessel-associated fibronectin may be only a function of adsorption onto basement membrane (type IV) collagen. To address these possibilities, we investigated the association of fibronectin with fibrin, type IV collagen, and mononuclear cell infiltrates in DH reactions. Skin sites of DH reactions in normal volunteers were biopsied at 24, 48, and 72 h after intradermal challenge and examined by immunofluorescence technique. At all time points most of the interstitial fibronectin coincided with fibrin; however, some interstitial fibronectin was coincident with mononuclear cells positive for HLA-DR or monocyte-specific antigen. The coincidence of fibronectin with mononuclear cells was more apparent in a 48-h DH reaction from a patient with congenital afibrinogenemia. Vessel wall fibronectin was increased by 48 h after challenge and appeared as a fine linear band on the luminal side of a much thicker band of type IV collagen. Thus, the coincidence of extravascular fibronectin with mononuclear cells, its appearance without fibrin in the site from a patient with afibrinogenemia, and incomplete correspondence of vessel wall fibronectin with type IV collagen suggest that fibronectin localization in DH reactions involves endothelial cell and mononuclear cell binding as well as adsorption to fibrin and/or type IV collagen.

Afibrinogenemia↗
PubMed1998

Fibronectin is a survival factor for differentiated osteoblasts.

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.

Animals↗
PubMed1984

Native and degraded fibronectin: new immunological methods for distinction.

Two methods for quantitative determination of the high molecular weight glycoprotein, fibronectin, have been developed. Both methods are based on enzyme-linked immunoadsorbent (ELISA) techniques. In the first of the methods an antibody against fibronectin is used to trap the antigen. This double antibody technique can detect a slight decrease in the concentration of fibronectin stored for 5 days compared to the amount of fibronectin in the freshly purified preparation. In the second method, gelatin which is known to bind specifically to fibronectin, is used to catch fibronectin. By this method less than 1% of the fibronectin present in a freshly prepared preparation is measured after storage for 5 days. The results obtained with the two methods applied on a freshly prepared and a stored fibronectin are in agreement with sodium dodecylsulphate polyacrylamide gel electrophoresis followed by immunoblotting before and after gelatin-Sepharose adsorption. These techniques demonstrate that all the freshly prepared fibronectin adsorbs to gelatin-Sepharose, while stored fibronectin, which is broken down to numerous peptides, still reacts with the fibronectin antibody, but does not adsorb to gelatin-Sepharose. The two ELISA techniques were applied on amniotic fluid, cerebrospinal fluid and urine. The results indicated significant degradation of fibronectin in urine, and less degradation of fibronectin in amniotic and spinal fluid.

Amniotic Fluid↗
PubMed1993

Chicken gonadotropin-releasing hormones enhance soluble and insoluble fibronectin production by granulosa cells of the domestic fowl in vitro.

Experiments were conducted in vitro to examine the effect of two chicken gonadotropin-releasing hormones, cGnRH-I ([Gln8]-GnRH) and cGnRH-II ([His5,Trp7,Tyr8]-GnRH), on fibronectin (soluble and insoluble) production by chicken granulosa cells isolated from the largest (F1; about 35 mm in diameter), and third largest (F3; 15 to 20 mm in diameter) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF; 6 to 8 mm in diameter). The amounts of soluble fibronectin (fibronectin secreted into the incubation medium) and insoluble fibronectin (fibronectin associated with cells plus fibronectin attached to culture substratum) were quantified with a specific ELISA. Fibronectin secreted into the incubation medium (soluble fibronectin) by unstimulated cells increased with advanced stages of follicular maturation. Addition of both cGnRH-I and -II increased the amount of fibronectin secreted into the incubation medium by all follicular cell types. The amount of insoluble fibronectin in culture wells that contained unstimulated cells also increased with advanced stages of follicle development. Both cGnRH-I and -II increased the quantity of insoluble fibronectin by granulosa cells from all follicle types. Total (soluble plus insoluble) fibronectin production was elevated when cGnRH-I or -II was added to F1, F3, and SYF granulosa cells. The magnitude of cGnRH-I or -II stimulation (percentage increase) of soluble, insoluble, or total fibronectin production was calculated as a multiple of the unstimulated (control) value for each follicle type, and they were greatest in cells derived from developing and immature follicles. These results indicate that homologous cGnRH-I and -II are capable of directly modulating the physiology of the avian ovary.

Animals↗
PubMed1989

Dual role of fibronectin in hematopoietic differentiation.

The adhesive glycoprotein fibronectin provides anchorage for fibroblasts and hematopoietic progenitor cells in vitro. Fibronectin also demonstrates growth factor activity for fibroblasts; however, there is no available information regarding its role as a hematopoietic growth factor. To distinguish growth factor activity of fibronectin from its anchorage activity for hematopoietic progenitors, we assessed the ability of purified human plasma fibronectin to promote human bone marrow erythroid, granulocyte-macrophage (GM) and mixed granulocyte-erythroid-macrophage-megakaryocyte (GEMM) colony formation in liquid suspension, methylcellulose, and fibrin clots under serum-free conditions. Addition of fibronectin to methylcellulose cultures, or to cultures formed in fibrin clots, using fibrinogen depleted of fibronectin by preadsorption over gelatin-Sepharose and clotted with thrombin, resulted in up to a twofold enhancement of proliferation of erythroid burst-forming units (BFU-E), erythroid colony-forming units (CFU-E), and CFU-GEMM. This effect was concentration-dependent up to a fibronectin supplement of 100 micrograms/mL. By contrast, CFU-GM proliferation was not affected by the addition of fibronectin to the cultures. Fibronectin-adherent marrow cells overlaid with liquid medium formed both early and late-appearing erythroid colonies, whereas similarly cultured plastic-adherent marrow cells did not. Erythroid colony formation was observed in cultures of fibronectin-adherent marrow cells overlaid with methylcellulose but not in cultures of plastic-adherent marrow cells under the same conditions. Finally, the erythroid growth-promoting activity of fibronectin was inhibited by arginyl-glycyl-aspartyl-serine (RGDS), a tetrapeptide that competitively blocks the interaction of fibronectin with its receptor. We conclude that fibronectin plays a dual role in hematopoiesis: providing (a) anchorage for erythroid and primitive progenitors, and (b) as a proliferative stimulus for these hematopoietic cells. Both activities may be mediated by the cell adhesion domain of the molecule.

Adult↗
PubMed1988

Kupffer cell complement receptor clearance function after surgical injury and phagocytosis of immune complexes: effect of changes in plasma fibronectin.

The present study evaluated the effect of changes in plasma fibronectin levels on the degree of depression of in vivo clearance function of Kupffer cell complement receptors after injury and the phagocytosis of immune complexes. In vitro studies have suggested that fibronectin may act as an opsonin for the clearance of immune complexes from the blood by binding to C1q and may influence the expression and activation of immune receptors on macrophages. Complement receptor clearance function was assessed in rats from the hepatic uptake of rat erythrocytes coated with antierythrocyte IgM. Increasing plasma fibronectin by the injection of purified fibronectin or decreasing fibronectin by the injection of gelatin had no effect on complement receptor function in otherwise normal animals. Surgical injury depressed both plasma fibronectin levels and complement receptor function. Injection of fibronectin at the time of injury prevented the depression of receptor function; however, when fibronectin was given 1 hour after injury, receptor function remained depressed even though fibronectin levels were increased. The phagocytosis of immune complexes (IgG-coated rat erythrocytes [EIgGs]) depressed complement receptor function but did not depress plasma fibronectin levels. The depression of receptor function caused by the phagocytosis of EIgGs was prevented by administering fibronectin and was potentiated by administering gelatin, which decreased plasma fibronectin levels. Therefore, plasma fibronectin concentrations can influence in vivo Kupffer cell complement receptor function under certain conditions that lead to the depression of complement receptor function.

Animals↗