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PubMed1995

Relationship between fibronectin expression during gastrulation and heart formation in the rat embryo.

By utilizing myosin immunostaining, we were able to identify early rat myocardium as a thin epithelial sheet and realized that its cohesive movement toward the midline leads to the straight heart tube formation. Localization study of fibronectin mRNA and protein was, therefore, carried out to investigate its tissue origin and possible roles in facilitating mesoderm migration and heart formation. Fibronectin mRNAs were first detected throughout the mesoderm during the early primitive streak stage, suggesting that the mesoderm is the source of fibronectin. By pre-head fold (pre-somite) and head fold (early somite) stages, the mesoderm became largely down-regulated for fibronectin mRNAs, while it was also at these stages when myosin-positive myocardium formed itself into the epithelium and was subsequently folding toward the midline. Thus, there appears to be little fibronectin synthesis during and directly relevant to early heart tube formation. Later, during the early straight heart tube stage (5 somite and older), endocardium became highly positive for fibronectin mRNAs, suggesting that the endocardium is the major source of fibronectin for the cardiac jelly. Based on the results, we present a map for the early mammalian heart in which the heart is a single crescentic band lying in front of the prechordal plate. We also suggest a process for heart tube formation based on the cohesive movement of the myocardial epithelium. During heart tube formation, fibronectin protein had been deposited previously by the mesoderm and was found uniformly in the ECM and not newly produced by any adjacent tissue. The data contradict the endodermal guidance of heart migration by fibronectin gradient and suggest, instead, a permissive role for the fibronectin substrate.

Animals↗
PubMed1983

Fibrin and fibronectin in rheumatoid synovial membrane and rheumatoid synovial fluid.

Normal synovial membranes and synovial membranes from patients with classic rheumatoid arthritis were investigated for the presence of fibrin and fibronectin by an indirect immunoperoxidase technique. In normal synovial membranes, fibronectin was found around the monolayer of the synovial lining cells. Staining was most intense on the surface and beneath the lining cells, but not detectable in the cytoplasm. Fibronectin was also found in the cytoplasm of the endothelial cells. No staining for fibrin was found in the normal synovial membrane. In synovial membranes from patients with rheumatoid arthritis, large amounts of fibronectin were found around the multilayer of synovial lining cells, in the cytoplasm of the endothelial cells, and in argyrophilic fiber-rich connective tissue. In superficial areas denuded of synovial lining cells, high amounts of fibronectin were found incorporated in fibrin. In some areas with noninjured synovial lining cells, fibrin was also found, but in this case no fibronectin was incorporated. No fibronectin was found in connective tissue in areas with infiltration of inflammatory cells. After treatment of normal and rheumatoid synovial membranes with hyaluronidase, fibronectin was still present around the lining cells but the staining was found to be more distinct. This study relates the presence of fibrin and fibronectin in the rheumatoid synovial membrane to the high amount of these proteins, recently described, in rheumatoid synovial fluid. It also suggests that fibronectin present in the synovial membrane is produced and secreted by the endothelial cells.

Arthritis, Rheumatoid↗
PubMed1986

Opsonic dysfunction secondary to plasma fibronectin depletion after aortic surgery.

Fibronectin is a plasma opsonin which is depleted after major injury or surgery. The effect of major aortic surgery on plasma fibronectin has been studied in patients and in an equivalent experimental model in pigs. On the day after aortic surgery in eleven patients mean fibronectin (+/- s.e.m.) had fallen from 363 +/- 11 mg/l pre-operatively to 179 +/- 19 mg/l (P less than 0.01). No such fall was observed in patients undergoing herniorrhaphy. In fourteen pigs aortic surgery produced reproducible surgical shock and a fall in plasma fibronectin from 331 +/- 10 mg/l to 43 +/- 13 mg/l after resuscitation (P less than 0.01). Whenever plasma fibronectin fell below 190 mg/l the circulating free fibronectin was consumed in complexes of 1000 kDa containing collagenous debris. More severe depletion of plasma fibronectin was related to higher concentrations of circulating nonopsonized collagenous debris and to subsequent mortality in pigs. The depletion of free fibronectin that occurs following major surgery may produce clinically important opsonic dysfunction. The clinical relevance of this fibronectin consumption may be missed if measurement is limited to circulating fibronectin levels without determining that proportion bound in complexes and no longer available as an opsonin.

Animals↗
PubMed2004

Radial glia produce and align the ligand fibronectin during neuronal migration in the developing chick brain.

We demonstrated previously that alpha8beta1 integrin regulates the migration and survival of immature neurons during development of the chicken optic tectum; however, the potential extracellular ligand was unknown. We used immunohistochemistry to determine if several potential ligands (fibronectin, tenascin, vitronectin, and osteopontin) were expressed during neuronal migration along radial glia (RG). Fibronectin was localized in a pattern relevant to radial migration and survival of neurons; it was present before and during neuronal migration and appeared oriented along RG fibers by conventional fluorescence microscopy. Confocal microscopy confirmed that fibronectin was localized along RG cells during radial migration. It was more concentrated in some superficial laminae, which might support directional movement. Fibronectin was present after formation of definitive tectal laminae, but was diffuse and not aligned along RG, which persist. Flow cytometry analysis of dissociated optic tectum cells revealed that almost all RG were positive for fibronectin. Short-term cell culture experiments using an exocytosis inhibitor revealed that fibronectin accumulated in most RG cells. Thus, fibronectin is produced by RG and is aligned along their surfaces before and during migration. Fibronectin, therefore, is a potential ligand for general radial neuronal migration in the chick optic tectum. Its predominant source appears to be RG, in contrast with developing mammalian cortex, where fibronectin was not found in a pattern that could guide widespread radial migration and where neurons are the predominant producers of fibronectin during migration.

Animals↗
PubMed1985

Fibronectin binding properties of bacteriologic petri plates and tissue culture dishes.

With the aid of a monoclonal antibody-based ELISA assay, the fibronectin binding properties of poly(styrene) bacteriologic and tissue culture petri plates were studied. After treatment of the plastics with serum, both the rate of fibronectin binding and the maximum amount of fibronectin bound were found to be lower for bacteriologic than tissue culture plates. In contrast, when treated with purified fibronectin rather than serum, bacteriologic and tissue culture plates bound fibronectin equally well. Thus, serum proteins are more effective in inhibiting fibronectin binding to bacteriologic petri plates than to tissue culture dishes. The fibronectin binding properties of plastic substrata could be enhanced by oxidation with H2SO4 or diminished by dissolution and recasting of tissue culture dishes. Thus, the fibronectin binding properties of bacteriologic and tissue culture dishes can be interconverted. Plastics with enhanced fibronectin binding properties (tissue culture plates) were found to be hydrophilic and good substrates for cell attachment and growth while plastics with decreased fibronectin binding characteristics were found to be hydrophobic and poor substrates for cell attachment and growth. The cell-adhesive properties of bacteriologic and tissue culture plastic substrata were found to vary during incubation with cells. While cells remained firmly attached and spread on tissue culture plastics over a period of 5 days or more, previously attached cells gradually detached from bacteriologic plastics at incubation times beyond 12 h. The gradual detachment of cells from bacteriologic plates probably explains the poor properties of bacteriologic plastics for the growth of anchorage-dependent cells, in particular.

Animals↗
PubMed1984

Fibronectin and wound healing.

I have tried to briefly review the evidence (summarized in Table II) indicating that fibronectin is important in cutaneous wound healing. Fibronectin appears to be an important factor throughout this process. It promotes the spreading of platelets at the site of injury, the adhesion and migration of neutrophils, monocytes, fibroblasts, and endothelial cells into the wound region, and the migration of epidermal cells through the granulation tissue. At the level of matrix synthesis, fibronectin appears to be involved both in the organization of the granulation tissue and basement membrane. In terms of tissue remodeling, fibronectin functions as a nonimmune opsonin for phagocytosis of debris by fibroblasts, keratinocytes, and under some circumstances, macrophages. Fibronectin also enhances the phagocytosis of immune-opsonized particles by monocytes, but whether this includes phagocytosis of bacteria remains to be determined. In general, phagocytosis of bacteria has not appeared to involve fibronectin. On the contrary, the presence of fibronectin in the wound bed may promote bacterial attachment and infection. Because of the ease of experimental manipulations, wound healing experiments have been carried out on skin more frequently than other tissues. As a result, the possible role of fibronectin has not been investigated thoroughly in the repair of internal organs and tissues. Nevertheless, it seems reasonable to speculate that fibronectin plays a central role in all wound healing situations. Finally, the wound healing problems of patients with severe factor XIII deficiencies may occur because of their inability to incorporate fibronectin into blood clots.

Animals↗
PubMed1983

Deposition of fibronectin on material surfaces exposed to plasma: quantitative and biological studies.

Experiments were carried out to characterize plasma fibronectin deposition onto material surfaces exposed to plasma solutions. Under nonclotting conditions, the amount of fibronectin adsorption on the surfaces, determined by an indirect radioactive antibody assay, was maximal at low plasma concentrations (0.1%). At higher concentrations of plasma, other plasma proteins appeared to compete with and inhibit adsorption of fibronectin. Biological activity (fibronectin-promoted cell spreading) was also greatest at low plasma concentrations and decreased as the plasma concentration was raised. When surfaces were exposed to plasma under clotting conditions (i.e., addition of Ca2+ and thrombin), fibronectin deposition on the surfaces and biological activity remained constant or increased as the plasma concentration was raised. Based on indirect immunofluorescent antibody assays, the fibronectin deposited from clotting plasma appeared to be in a punctate distribution over the entire material surface and occasionally was associated with discrete fibrillar structures. The increased deposition of fibronectin from clotting plasma compared to nonclotting plasma (approximately a 10-fold difference with 10% plasma) was partially a result of covalent crosslinking of fibronectin to fibrin based upon studies with putrescine added to inhibit crosslinking during clotting. On the other hand, the increase in biological activity that occurred if the surfaces were exposed to clotting plasma was completely inhibited by putrescine, indicating that fibronectin had to be crosslinked to fibrin to have biological activity under these conditions. Finally, fibronectin deposition also occurred on surfaces exposed to whole blood and was markedly enhanced when clotting occurred.

Adsorption↗
PubMed1995

Reciprocal induction of tumor necrosis factor-alpha and interleukin-1 beta activity mediates fibronectin synthesis in coronary artery smooth muscle cells.

We previously demonstrated an immune-inflammatory response associated with increased expression of interleukin (IL)-1 beta and fibronectin in graft coronary arteriopathy in piglets following heterotopic heart transplant. Further studies showed that increased endogenously produced IL-1 beta was upregulating fibronectin production by donor coronary artery (CA) smooth muscle cells (SMC). Since co-induction of IL-1 beta and tumor necrosis factor (TNF)-alpha has been shown in other systems, we investigated the possible interaction between these cytokines in regulating fibronectin production in CA SMC. First, we documented increased TNF-alpha expression in vivo in donor compared to host CA. Next, synthesis of fibronectin was measured in host and donor CA SMC following [35S]-methionine radiolabeling and gelatin-sepharose extraction. As previously shown with IL-1 beta, increased donor CA SMC fibronectin synthesis was reduced to host levels in the presence of TNF-alpha antibodies, and exogenous TNF-alpha upregulated fibronectin synthesis in host CA SMC to levels in donor cells. In normal CA SMC, TNF-alpha-stimulated fibronectin production was downregulated to or below control levels in the presence of IL-1 beta antibodies. Likewise, IL-1 beta-stimulated fibronectin synthesis was downregulated to control levels when TNF-alpha neutralizing antibodies were added. Combining TNF-alpha and IL-1 beta enhanced fibronectin production over that observed with either cytokine alone, but was not additive. Thus, our studies suggest that vascular SMC fibronectin synthesis is regulated by reciprocal induction of IL-1 beta and TNF-alpha activity and provide the first demonstration of a 'cytokine loop' modulating matrix production.

Animals↗
PubMed1991

Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase.

Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (less than 45 kD), derived from the artificial matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.

Amino Acid Chloromethyl Ketones↗
PubMed1993

Hepatic removal of 125I-DLT gelatin after burn injury: a model of soluble collagenous debris that interacts with plasma fibronectin.

The decline of plasma fibronectin after surgery, trauma, and burn, as well as during severe sepsis after injury, appears to limit hepatic Kupffer cell phagocytic activity. Intravenous infusion of gelatin-coated particles to simulate blood-borne particulate collagenous tissue debris in the circulation after injury also depletes plasma fibronectin. We used soluble gelatin conjugated with 125I-labeled dilactitol tyramine (DLT-gelatin) as a model of soluble collagenous tissue debris. We studied its blood clearance as well as organ localization in normal and postburn rats. Fibronectin-deficient plasma harvested early after burn exhibited limited ability to support in vitro phagocytic uptake of the gelatinized microparticles by Kupffer cells in liver tissue from normal rats. However, Kupffer cells in liver tissue from normal and postburn rats phagocytized the test particles at a normal rate when incubated in normal plasma. The DLT-gelatin ligand bound to fibronectin in a dose-dependent manner as verified by its capture with anti-fibronectin coated plastic wells when coincubated with purified fibronectin. By gel filtration chromatography, the binding of fibronectin with the DLT-gelatin ligand was readily detected, resulting in the formation of a high-molecular-weight complex. In normal animals the plasma clearance and liver localization of 125I-DLT-gelatin was competitively inhibited by infusion of excess nonradioactive gelatin. The blood clearance and liver localization of the soluble gelatin ligand were also impaired after burn injury during periods of fibronectin deficiency similarly to the pattern observed with gelatin-coated microparticles. By autoradiography, the cellular site for the uptake of the 125I-DLT-gelatin was primarily but not exclusively hepatic Kupffer cells; 125I-DLT-asialofetuin and 125I-DLT-ovalbumin were removed by hepatocytes and sinusoidal endothelial cells, respectively. Thus, gelatin conjugated with 125I-DLT can be used to simulate blood-borne soluble collagenous tissue debris after burn. It rapidly binds to plasma fibronectin before its hepatic Kupffer cell removal, and its blood clearance is markedly delayed after burn injury during periods of plasma fibronectin deficiency.

Animals↗
PubMed1995

Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth.

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits alpha 3, alpha 5, alpha IIb, alpha v, beta 1 and beta 3, but not alpha 4, all bound to trophoblast cells. Antibodies raised against either the beta 1 or beta 3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, alpha 3 beta 1, alpha 5 beta 1, alpha IIb beta 3, and alpha v beta 3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation.

Amino Acid Sequence↗
PubMed1993

Evidence for a glycosaminoglycan chain on a portion of articular cartilage fibronectins.

Fibronectin heterogeneity is, in part, the result of post-translational modifications. In these experiments, cartilage fibronectins were purified by anion exchange chromatography, followed by gelatin affinity chromatography or immunoprecipitation, and, finally, sodium dodecyl sulfate--polyacrylamide gel electrophoresis (NaDodSO4 PAGE). A substantial, although variable, portion of the fibronectins from canine and equine cartilages of all ages required salt concentrations from 0.2 to 1.0 M for elution from DEAE-cellulose. This was in contrast to plasma fibronectin which eluted with 0.1 M NaCl, but these results were consistent with observations made on human cartilage by Brown and Jones (1990 J. Rheumatol. 17, 65-72). When cartilage explants were incubated with Na2 35SO4=, the cartilage fibronectins were sulfated and the fibronectins which eluted with high salt contained from 5- to 50-fold more radiosulfate than the fibronectins which eluted with 0.1 M NaCl. A fraction of the 35SO4= which copurified with the cartilage fibronectin and comigrated with it in NaDodSO4-PAGE could be removed by digestion with chondroitinase ABC. This suggested that a percentage of cartilage fibronectins are covalently linked to a chondroitin sulfate or dermatan sulfate chain and thus might also appropriately be called proteoglycans. Alternatively, there is a proteoglycan which binds so tightly to fibronectin that separation is not achieved even in the presence of urea, sodium dodecyl sulfate, and mercaptoethanol.

Animals↗
PubMed1996

Fibronectin fibrils and growth factors stimulate anchorage-independent growth of a murine mammary carcinoma.

Stromal cells are important regulators of mammary carcinoma growth and metastasis. We have previously shown that a 3T3-L1 adipocyte cell line secretes hepatocyte growth factor (HGF), which stimulates proliferation of a murine mammary carcinoma (SP1) in monolayer cultures (DNA Cell Biol. 13, 1189-1897, 1994). We now examine the role of growth factors and the extracellular matrix protein fibronectin in stimulation of anchorage-independent growth of SP1 cells. Purified transforming growth factor-beta (TGF-beta) stimulated significant colony growth in soft agar cultures, whereas HGF had a lesser effect. Analysis by confocal microscopy revealed that carcinoma cell colonies contained extracellular microfibrils composed of fibronectin. Partial depletion of fibronectin from 7% FBS/agar cultures reduced the number of colonies; colony growth could be recovered by adding back exogenous fibronectin. Addition of the 70-kDa N-terminal fragment of fibronectin, which inhibits fibronectin fibril formation, reduced growth of SP1 cell colonies, but an 85-kDa fragment containing the cell binding domain did not inhibit colony growth. These findings indicate that deposition of extracellular fibronectin fibrils is necessary, but not sufficient, for anchorage-independent growth of SP1 mammary carcinoma cells; growth factors are also required. SP1 cells had less fibronectin mRNA and secreted less fibronectin protein under anchorage-independent conditions than under anchorage-dependent conditions, as determined by Northern blotting and immunoprecipitation analysis. Thus, both growth factors (HGF and TGF-beta) and fibronectin may be important regulators of paracrine stimulation by stromal cells of anchorage-independent growth of mammary carcinoma cells.

3T3 Cells↗
PubMed1997

Identification and characterization of a new protein from Streptococcus pyogenes having homology with fibronectin and fibrinogen binding proteins.

Fibronectin and fibrinogen-binding proteins have been described as possible adhesin in streptococci and staphylococci. Recent published data has demonstrated that Protein F, a fibronectin-binding protein from group A streptococci, is important in adherence to respiratory cells (8). Other similar proteins already described (i.e. SOF, Sfb and SfbII) are able to competitively inhibit the binding of fibronectin to S. pyogenes (9,10,5). Similarly, clumping factor from S. aureus, is known to promote adherence to fibrinogen-coated surfaces (7). When the sequence from SFFBP-12 was compared against all the others fibronectin and fibrinogen-binding proteins described in streptococci and staphylococci (1-10), an identity at the amino acid level, ranging from 38 to 69% was found for the C region. For the repeated region (R), the identity ranged between 47 and 75%. Unlike all the other proteins already described in group A streptococci, the protein we describe here, SFFBP-12, shares a high degree homology (67-75%) with the fibronectin-binding protein B from S. dysgalactiae (6), as well as homology with the S. aureus clumping factor (7) and fibronectin-binding protein B (3), making it a new potential fibronectin-fibrinogen binding protein for group A streptococci. These characteristics would also imply that SFFBP-12 contains two different fibronectin-binding domains (regions B and C), thus enhancing its role as a possible major adhesin molecule. RNA transcription assays showed a transcript with the expected molecular size for the intact SFFBP-12 protein, confirming that the protein is actively expressed during bacterial growth. SFFBP-12 is the largest protein of its kind identified from group A streptococci and is comparable in size to the fibronectin binding protein B from S. dysgalactiae (6). If it is shown that SFFBP-12 does in fact bind both fibronectin and fibrinogen, as the sequence data suggest, it would make this molecule a major virulence determinant for the group A streptococcus.

Adhesins, Bacterial↗
PubMed1986

Plasma fibronectin: relevance for anesthesiology and intensive care.

Plasma fibronectin has been postulated to be an essential mediator of normal reticuloendothelial system (RES) function. The acute depletion of fibronectin is thought to impair RES function, whereas its repletion in states of deficiency has been reported to improve RES function. In vitro studies have documented fibronectin's ability to bind to some nonbacterial microaggregates and to promote the phagocytosis of bound targets by the RES. These properties may, however, be influenced by the in vivo milieu. There is substantial evidence for a parallelism between RES function and plasma fibronectin levels following blunt trauma in animal models; however, this association is not seen in experimentally induced intravascular coagulation, acute inflammation, and sepsis. Clinically, subnormal fibronectin levels are clearly associated with the triad of intravascular coagulation, organ failure and sepsis. Fibronectin is, however, not the only plasma protein reduced in these patients, nor is it an outstanding predictor of such complications. The therapeutic efficacy of fibronectin administration remains controversial. Whereas initial reports suggested therapeutic benefits of fibronectin-enriched cryoprecipitates, subsequent studies have produced negative results. Prospective, randomized, controlled clinical trials with purified fibronectin are needed before fibronectin should be recommended as an adjunct to the established principles of intensive care.

Anesthesia↗
PubMed1982

Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique.

Fresh frozen tissue sections of human articular cartilage was treated without and with human testicular hyaluronidase (2 x 10(6) units/l) for 60 min at 37 degrees C and stained by the indirect immunoperoxidase technique with rabbit antihuman fibronectin. The rabbit antihuman fibronectin was purified by affinity chromatography on human fibronectin-Sepharose. Fibronectin was only found on the acellular surface of the articular cartilage in tissue sections not treated with hyaluronidase. In this surface layer, probably identical to "lamina splendens", the arrangement of fibronectin was as a membrane. No collagen was seen in this area by van Gieson staining. No staining for fibronectin was found in the cartilage matrix or in the chondrocytes. Treatment of the cartilage tissue with hyaluronidase resulted in visualization of high amount of fibronectin in the cartilage matrix, with the highest intensity around the chondrocytes. The staining of the acellular surface layer of the articular cartilage was identical with the results obtained without hyaluronidase treatment. These results indicate that articular cartilage is rich in fibronectin probably in complex with hyaluronic acid, and that the chondrocytes produce fibronectin in situ. It also demonstrates the steric hindrance of hyaluronic acid aggregates in diffusion of the antibody and the value of hyaluronidase treatment of tissue before demonstration of fibronectin.

Cartilage, Articular↗
PubMed1991

Leukocyte elastase-independent proteolysis of gelatin-bound fibronectin by inflammatory macrophages.

Fragmentation of lung matrix fibronectin by proteases released from activated phagocytic cells has been implicated in lung vascular injury. We examined whether denatured collagen (gelatin)-bound fibronectin can be degraded by peritoneal exudate mononuclear phagocytes harvested from rats 96 h after intraperitoneal casein injection. Microtiter plates were pretreated with gelatin and then supplemented with purified 125I rat plasma fibronectin, which readily bound to the gelatin. Stimulated inflammatory exudate cells were added and proteolysis of the bound fibronectin was studied by the release of [125I]fibronectin fragments into the media. Following 2 h of incubation, peritoneal exudate mononuclear macrophages stimulated with opsonized zymosan released three times more radiolabeled fibronectin into the medium as compared to background controls, and 1.5 times more radiolabeled fibronectin as compared to cells not stimulated with zymosan. Western blot analysis and autoradiography confirmed the presence of fragments of fibronectin in the culture medium. Some of these fragments were clearly derived from the radiolabeled matrix, but others that were not labeled were potentially released directly from the added stimulated macrophages. The release of radiolabeled fibronectin was inhibited by N-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin specific inhibitor, but not by methoxysuccinyl-alanine-alanine-proline-valine-chloromethyl-ketone (AAPVCK), a leukocyte elastase-specific inhibitor. These results suggest that fibronectin bound to denatured collagen is susceptible to leukocyte elastase-independent enzymatic degradation by stimulated inflammatory exudate mononuclear phagocytic cells. Such proteolysis may mimic a pathological process associated with lung vascular injury during the sequestration of activated macrophages in the lung microcirculation and interstitium.

Animals↗
PubMed1990

A study on content and distribution of plasma and tissue fibronectin in rats using ELISA and immunofluorescence.

The content of plasma fibronectin and tissue extractable fibronectin in normal rats was measured with rocket immunoelectrophoresis and ELISA in the present study. The difference in tissue fibronectin distribution in various organs and the correlation between distribution and content of fibronectin have been studied. We suggest that tissue fibronectin may be a complex component. The change in plasma fibronectin reflects a dynamic balance existing between the tissue fibronectin pool and plasma fibronectin pool in normal rats. Plasma fibronectin and tissue fibronectin concentrations are not static, and they can only be maintained in a relatively stable state in normal rats.

Animals↗