Hydrodynamics of cryogelation

Cryogel, prevalent in the plasma of rheumatoid arthritis patients, is a plasma fibronectin (pFN)-extra domain A containing FN [EDA(+)FN]-fibrinogen (Fbg) aggregate formed by the addition of heparin (Hep) at low temperature. Although EDA(+)FN is not usually present in normal plasma, its prevalence in rheumatic patients induces cryogelation. In this study, we determined the hydrodynamic radius (R_h) ratio (R_h/R_h30) of the cryogel component by dynamic light scattering in vitro. R_h/R_h30 was normalized to R_h at 30 °C (R_h30) at several temperatures. The R_h/R_h30 of Fbg was found to increase only by self-aggregation, whereas the R_h/R_h30 of FNs does not increase in response to temperature changes. The R_h/R_h30 of the Fbg/FN aggregate is increased by the addition of Hep, and the R_h/R_h30 (12.5) of the Hep-induced EDA(+)FN/Fbg aggregate is greater than that (2.5) of the pFN/Fbg aggregate. These results suggest that cryogelation requires Fbg self-aggregation and the interaction between EDA(+)FN and Hep.     

Development of a novel polycationic adsorbent for cryogel removal.

Cryogel, prevalent in the plasma of rheumatism patients, is a plasma fibronectin (pFN)-extra domain A containing FN (EDA(+)FN)-fibrinogen (Fbg) complex formed by adding heparin (HP) at a low temperature (4 degrees C). Although EDA(+)FN does not usually exist in normal plasma, its prevalence in rheumatic patients causes cryogelation in plasma. Removal of cryogel is thus a promising and novel approach to treating rheumatism. As HP-EDA(+)FN aggregate, which is induced by the main component of cryogel, is considered to be an anion, cationic materials capable of eliminating this anionic conjugate were innovated in this study. We found that an amino group density of 100-130 micromol/g (dry weight) of adsorbents prompted selective adsorption of the EDA(+)FN-HP complex. Elimination of EDA(+)FN as high as 80% accompanied by removal of the components of total FN (pFN) (10%) and Fbg (10%) in the model patient plasma was established.     

Inhibition of cryogelation by the novel synthetic peptide (Gly-Arg-Lys-Lys-Thr): recognition site of extra domain A containing fibronectin for heparin in cryogelation

Cryogel is a physical gel formed by the heterophilic aggregation of extra domain A (EDA) containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep) in the blood of rheumatoid arthritis (RA) patients. In cryogelation EDA(+)FN cross-links to form an interaggregate of cryogel with Hep. In the present study, we determined the recognition structure of Hep for EDA(+)FN by using oligo- and desulfonated-Hep. The affinity constant (KA) (1.2×10⁸ per M) of oligo-Hep for EDA(+)FN did not change with a decrease in number-average molecular weight (4.9×10⁴→6.0×10³). The KA-value of desulfonated-Hep for EDA(+)FN decreased from 3.2×10⁸ to 1.0×10⁷ per M with a decrease in the sulfonation ratio (7.0→4.3%). We also determined the recognition structure of EDA(+)FN for Hep by an inhibition experiment on the heparin binding domain II (HepII) in EDA(+)FN with the synthetic peptides, Arg–Arg–Ala–Arg (RRAR), Asp–Gln–Ala–Arg (DNAR), Ile–Lys–Tyr–Glu–Lys (IKYEK), and Gly–Arg–Lys–Lys–Try (GRKKT). The GRKKT sequence clearly inhibited bonding between EDA(+)FN and Heps containing oligo- and desulfonated-Hep. The amount of cryogel formed in the RA-patient model plasma corresponded to the EDA(+)FN concentration in cryogel (36.7%) normalized by the EDA(+)FN concentration in plasma. When GRKKT was added to plasma, the EDA(+)FN concentration fell to 10.5%. These results demonstrated that inhibition of cryogelation in plasma could progress to a novel treatment for RA.     

Specific interactions between cryogel components: role of extra domain A containing fibronectin in cryogelation

Cryogel is a physical gel formed by heterophilic aggregation of extra domain A containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep), which are found in high concentrations in the blood of patients suffering from rheumatoid arthritis. In this study, we clarify the specific interactions between cryogel components in terms of the affinity constant (K(A)), obtained by surface plasmon resonance (SPR). It is found that Fbg self-interactions occur at lower temperatures, and that K(A) of Fbg-Hep changes with temperature. Specifically, K(A) (2.0 x 10(8) [M(-1)]) of Fbg-Hep at 5 degrees C increases significantly from that (1.0x10(7) [M(-1)]) at 40 degrees C. K(A) of EDA(+)FN-Hep increases with temperature, by approximately 100-fold between 40 degrees C (K(A)=10(12) [M(-1)]) and 20 degrees C (K(A)=10(10) [M(-1)]). Although K(A) of the FN fragments of Hep-binding domain containing an EDA region [EDA(+)HBD(+)] and Hep increases with temperatures above 30 degrees C, K(A)s of HBD(+)-Hep and EDA(+)-Hep are not temperature-dependent. Therefore, EDA(+)HBD(+), formed as a special structure for high Hep affinity, exhibits temperature-dependent interaction with Hep. These results suggest that the main role of EDA(+)FN in cryogelation is to support the interaction with Hep.     

The alternatively spliced V region contributes to the differential incorporation of plasma and cellular fibronectins into fibrin clots

During blood clot formation in vivo, plasma fibronectin (pFN) is cross-linked to fibrin by coagulation factor XIIIa. Cellular FN (cFN), which localizes to connective tissue, is distinguished from pFN by the inclusion of alternatively spliced segments. To determine if these two FNs are functionally equivalent in blood clotting, the cross-linking of rat pFN and cFN to fibrin was compared in an in vitro clotting assay. Fibrinogen and FN were incubated at physiological ratios in the presence of thrombin and factor XIIIa. Cross-linking of FN to fibrin was monitored by SDS-PAGE and immunoblotting. Over 24 h, cFN was incorporated at a significantly slower rate than pFN and was not completely cross-linked to fibrin at a temperature that favors this interaction (0 degrees C). This difference was observed with purified fibrinogens from human, rat, and bovine and with rat plasma and was maintained even after incubation of pFN with rat fibroblasts for several days. Using the same assay, purified recombinant V(+)-V0 and V(+)-V+ FN dimers resembling pFN and cFN, respectively, showed a similar difference in cross-linking kinetics. These results suggest that the asymmetric distribution of the V region among pFN dimers plays a role in regulating its incorporation into blood clots. In fibrin clots, cFN was converted into a set of cross-linked intermediates distinct from those of pFN. For example, while pFN was initially cross-linked into a pFN-fibrin alpha heterodimer, this product was not a major intermediate in clots formed with cFN. This finding, in conjunction with evidence for the formation of factor XIIIa-catalyzed cFN-cFN cross-links, indicated that cFN molecules interact with each other, and with fibrin, differently from pFN. Together, these results show an important functional distinction between pFN and cFN.     

Preparation of gellan sulfate as an artificial ligand for removal of extra domain A containing fibronectin

The extra domain A containing fibronectin (EDA(+)FN) concentration in plasma of rheumatoid arthritis (RA) is abnormally higher than the normal level. We synthesized various gellan-sulfate (GS) candidates as artificial ligands for removing EDA(+)FN from plasma. The interaction between these artificial ligands and EDA(+)FN was evaluated using affinity constants (KA), which were determined by surface plasmon resonance measurement. The KA (3.6×10⁸ per M) of GS-25 [degree of substitution for sulfonation (DS)=25%] with EDA(+)FN was higher than those of other molecules: GS-16 (DS=16%) at 8.3×10⁷ per M, and GS-35 (DS=35%) at 1.7×10⁸ per M. Furthermore, GSs displayed selectivity of EDA(+)FN for binding with plasma FN (KA_EDA(+)FN/KA_{plasma FN}>2). The removal ratio in plasma was measured by using GS-immobilized gel. Removals of 66, 11, 7.7, 6.2, 6.9, and 12% for EDA(+)FN, plasma FN, fibrinogen, albumin, immunoglobulin G (IgG) and antithrombin III from the patient-model plasma were, respectively, achieved with GS-25-immobilized gel. These results suggest that GS may be used as a selective artificial ligand for EDA(+)FN removal from plasma in RA treatment.     

Immobilized gellan sulfate surface for cell adhesion and multiplication: development of cell-hybrid biomaterials using self-produced fibronectin

A new concept for cell-hybrid biomaterial is proposed in which human unbilical vein endothelial cells (HUVEC) are adhered to an immobilized gellan sulfate (GS) surface. Extra domain A containing fibronectin (EDA(+)FN) released from HUVEC is necessary for cell adhesion and multiplication. The material design in this study is based on these self-released cell adhesion proteins. The interaction between GS and EDA(+)FN was evaluated using the affinity constant (KA); the value obtained was 1.03x10(8) (M(-1)). These results suggest that the adhesion of HUVEC to GS may be supported by the adhesion of EDA(+)FN to GS. We also found that this new material adheres to HUVEC, allowing the reintroduction of EDA(+)FN, which is self-produced by the cell. This material is relatively easy to produce, not requiring the usual coating of adhesion proteins in pretreatment.     

Extra domain A-containing fibronectin expression in Spin90-deficient fibroblasts mediates cancer-stroma interaction and promotes breast cancer progression

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play major roles in supporting cancer progression. A previous report showed that SPIN90 downregulation is correlated with CAF activation and that SPIN90-deficient CAFs promote breast cancer progression. However, the mechanisms that mediate cancer-stroma interaction and how such interactions regulate cancer progression are not well understood. Here, we show that extra domain A (EDA)-containing fibronectin (FN), FN(+)EDA, produced by mouse embryonic fibroblasts (MEFs) derived from Spin90-knockout (KO) mice increases their own myofibroblast differentiation, which facilitates breast cancer progression. Increased FN(+)EDA in Spin90-KO MEFs promoted fibril formation in the extracellular matrix (ECM) and specifically interacted with integrin α4β1 as the mediating receptor. Moreover, FN(+)EDA expression by Spin90-KO MEFs increased proliferation, migration, and invasion of breast cancer cells. Irigenin, a specific inhibitor of the interaction between integrin α4β1 and FN(+)EDA, significantly blocked the effects of FN(+)EDA, such as fibril formation by Spin90-KO MEFs and proliferation, migration, and invasion of breast cancer cells. In orthotopic breast cancer mouse models, irigenin injection remarkably reduced tumor growth and lung metastases. It was supported by that FN(+)EDA in assembled fibrils was accumulated in cancer stroma of human breast cancer patients in which SPIN90 expression was downregulated. Our data suggest that SPIN90 downregulation increases FN(+)EDA and promotes ECM stiffening in breast cancer stroma through an assembly of long FN(+)EDA-rich fibrils; moreover, engagement of the Integrin α4β1 receptor facilitates breast cancer progression. Inhibitory effects of irigenin on tumor growth and metastasis suggest the potential of this agent as an anticancer therapeutic.     

Differential effects of cellular fibronectin and plasma fibronectin on ovarian cancer cell adhesion, migration, and invasion

Summary The main form of fibronectin (FN) encountered by tumor cells in vivo is cellular FN (cFN), which differs structurally and functionally from the commonly used plasma FN (pFN). We compared the effects of cFN and pFN on the ovarian carcinoma lines OVCAR-3 and SKOV-3 and on cultures of normal ovarian surface epithelium, which is the precursor of the epithelial ovarian carcinomas. Ovarian surface epithelial cells and SKOV-3 cells attached and spread faster on cFN than on pFN. On cFN, SKOV-3 migration was enhanced compared with pFN or plastic. In a matrigel transfilter assay, cFN strongly inhibited SKOV-3 invasion, whereas pFN did not. In contrast to SKOV-3, OVCAR-3 cells adhered faster on FN than on plastic but did not discriminate between cFN and pFN, and they did not migrate or invade matrigel either with or without FN. In both carcinoma lines, proliferation was unaffected by either FN. The results show profound differences in the responses to cFN and pFN by two invasive ovarian carcinoma lines. Because cFN is the main type that cancer cells encounter in vivo, extrapolations from culture data to in vivo events should preferably be based on studies using this form of FN.     

Novel plasma-separation dilayer gellan-gellan-sulfate adsorber for direct removal of extra domain A containing fibronectin from the blood of rheumatoid arthritis patients

Rheumatoid arthritis (RA) patients, in whom cryogelation occurs in the presence of heparin, exhibit abnormally high concentrations of extra domain A containing fibronectin [EDA(+)FN] in their plasma. The selective removal of EDA(+)FN from patient blood is therefore of potential therapeutic benefit. Gellan-sulfate is a candidate ligand for the removal of EDA(+)FN due to its high affinity for FN. In this study, we prepare a novel adsorber for the direct removal of EDA(+)FN from patient blood. The adsorber has both a plasma separation function and EDA(+)FN trapping zones, and is prepared by cross-linking gellan-sulfate with epichlorohydrine. The ratio of gellan-sulfate to gellan in the adsorber is 48%. The surface and internal structure of gellan beads were observed by a range of microscopic techniques, and the beads were found to have a dilayer structure, consisting of a porous outer layer and an underlying gellan-sulfate phase as the adsorber. The affinity constants of the gellan-sulfate beads for EDA(+)FN were almost the same in blood as in buffer because the porous gellan coating acts to separate plasma from the cellular fraction of the blood. The removal rate of plasma proteins and blood cells from mock RA blood was measured for coated and uncoated gellan-sulfate beads. Removal rates were 30-32% for EDA(+)FN, 6-10% for fibrinogen, 10-14% for antithrombin III, 8% for C3, 4-7% for C4, and 0% for albumin. The removal rates of uncoated beads were 11% for white blood cells, 0% for red blood cells and 33% for platelets, whereas removal rates of 0% for white blood cells, 0% for red blood cells and 20% for platelets were achieved for coated beads. The coating effectively inhibits the adsorption of white blood cells and platelets. Existing problems with direct adsorbers, including selectivity and plasma separation, have been solved by this material.     

Assembly of exogenous fibronectin by fibronectin-null cells is dependent on the adhesive substrate

The role of endogenously synthesized fibronectin (FN) in assembly was studied with cells lacking or expressing FN. Cells were cultured as homogeneous or mixed populations on surfaces coated with different matrix proteins. Compared with FN-expressing cells, FN-null cells poorly assembled exogenous plasma FN (pFN) when adhered to vitronectin or the recombinant cell-binding domain (III(7-10)) of FN. Vitronectin had a suppressive effect that was overcome by co-adsorbed pFN or laminin-1 but not by soluble FN. In co-cultures of FN-expressing cells and FN-null cells, endogenous FN was preferentially assembled around FN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was vitronectin, exogenous pFN assembled preferentially around cells expressing cellular FN or recombinant EDa- or EDa+ FN. In co-cultures on vitronectin of FN-null cells and beta(1) integrin subunit-null cells, fibrils of cellular FN and pFN were preferentially deposited by FN-null (beta(1)-expressing) cells immediately adjacent to (FN-secreting) beta(1)-null cells. In co-cultures on vitronectin of FN-null cells and beta(1)-null cells expressing a chimera with the extracellular domain of beta(1) and the cytoplasmic domain of beta(3), preferential assembly was by the chimera-expressing cells. These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN (suppressive if vitronectin, non-suppressive but non-supportive if III(7-10), supportive if pFN or laminin-1) and suggest that efficient interaction of freshly secreted cellular FN with a beta(1) integrin, presumably alpha(5)beta(1), substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or pFN to support assembly of FN.     

Pioglitazone attenuates TGF-beta(1)-induction of fibronectin synthesis and its splicing variant in human mesangial cells via activation of peroxisome proliferator-activated receptor (PPAR)gamma

The peroxisome proliferator-activated receptor (PPAR)gamma is expressed not only in adipose tissue but also in macrophages/monocytes and plays important roles in acute/chronic inflammation. Transforming growth factor (TGF)-beta is a common pathogenic indicator of sclerosis because it induces the accumulation of extracellular matrix (ECM) in the glomerular mesangium of the kidney. Among components of the ECM, fibronectin (FN) is an acute reactant in inflammation, and isoforms of it produced by splicing of gene variants appear during abnormal conditions such as wound healing. In this study, we examined the effects of pioglitazone, a PPARgamma agonist, on TGF-beta(1)-induced FN synthesis in cultured mesangial cells using RT-PCR and Western blot analysis. We also analyzed its splicing variant, extra domain (ED) A, containing FN (EDA(+)FN). TGF-beta(1) enhanced the production of both FN and EDA(+) FN and down-regulated PPARgamma expression. Pioglitazone reversed both these effects of TGF-beta(1). These findings suggest that PPARgamma activation by pioglitazone may affect the TGF-beta(1)-induced FN accumulation observed in the glomerular mesangium in cases of glomerulosclerosis, although further in vivo experiments are needed to evaluate this inference.     

Regulation of fibronectin expression and splicing in migrating epithelial cells: migrating MDCK cells produce a lesser amount of, but more active, fibronectin

Previously we have demonstrated that in MDCK epithelial cells not only transforming growth factor-beta (TGF-beta) but also hepatocyte growth factor/scatter factor (HGF/SF) regulates fibronectin (FN) splicing by increasing the ratio of EDA-containing FN (EDA+ FN) mRNA to EDA-minus FN (EDA- FN) mRNA (EDA+/EDA- ratio). EDA+ FN is known to be upregulated in tissues where cells actively migrate, such as those during morphogenesis, wound healing, and tumorigenesis. However, a direct association between cell migration and FN splicing at the EDA region has never been investigated. In this work, we have shown by using an in vitro wound migration assay that migrating epithelial cells regulate FN production and splicing differently compared to nonmigrating cells. Wounds were introduced as migration stimuli into the 10-day-old confluent cell sheet, where the EDA+/EDA- ratio and FN mRNA expression levels were stable. In migrating cells at the wound edge, the FN mRNA level decreased by 0.73-fold and the EDA+/EDA- ratio increased by 1.32-fold when compared with nonmigrating cells apart from the wound edge. HGF/SF significantly stimulated cell migration at the wound edge and concomitantly decreased the FN mRNA level by 0.60-fold and increased the EDA+/EDA- ratio by 1.84-fold in migrating cells. In nonmigrating cells apart from the wound edge, FN mRNA expression and splicing were not influenced by either wound stimulation or HGF/SF. EDA+ FN stimulates cell migration more effectively than EDA- FN and thus is considered to be a more active variant of FN. Taken together, migrating MDCK cells appear to regulate FN mRNA expression and splicing to produce a lesser amount of, but more active, FN.     

Hepatocyte growth Factor/Scatter factor (HGF/SF) is a regulator of fibronectin splicing in MDCK cells: comparison between the effects of HGF/SF and TGF-beta1 on fibronectin splicing at the EDA region.

EDA-containing fibronectin (EDA + FN) is selectively produced under several physiological and pathological conditions requiring tissue remodeling, where cells actively proliferate and migrate. Only a few growth factors, such as transforming growth factor (TGF)-beta1, have been reported to regulate FN splicing at the EDA region. In the present study, we showed for the first time that hepatocyte growth factor/scatter factor (HGF/SF), which is mainly produced by mesenchymal cells and functions as a motogenic and mitogenic factor for epithelial cells, modulates FN splicing at the EDA region in MDCK epithelial cells. HGF/SF treatment increased the ratio of EDA + FN mRNA to mRNA of FN that lacks EDA (EDA - FN) (EDA+/EDA- ratio) more than TGF-beta1 treatment did: at a range from 0.02 to 20 ng/ml, HGF/SF increased the ratio in a dose-dependent manner by up to 2. 1-fold compared with nontreated control, while TGF-beta1 stimulated the EDA+/EDA- ratio by 1.5-fold at the optimum dose of 10 ng/ml. However, TGF-beta1 increased total FN mRNA levels by 3-fold at 10 ng/ml, but HGF/SF did not. We previously demonstrated that fibroblasts cultured at low cell density expressed more EDA + FN than those at high cell density. The same effect of cell density was also observed in MDCK cells. Furthermore, at low cell density, HGF/SF stimulated EDA inclusion into FN mRNA more effectively than did TGF-beta1, whereas at high cell density, TGF-beta1 was more potent than HGF/SF. Simultaneous treatment of cells with HGF/SF and TGF-beta1 synergistically stimulated EDA inclusion into FN mRNA. This stimulation of EDA inclusion into FN mRNA by HGF/SF led to increased EDA + FN protein production and secretion by cells, which was demonstrated by immunoblotting. Thus, our studies have shown that HGF/SF is an enhancer of EDA inclusion into FN mRNA as is TGF-beta1. However, these two factors were different in their effects at low and high cell densities and also in their effects on total FN mRNA levels.     

The proteolytic activity of the recombinant cryptic human fibronectin type IV collagenase from E. coli expression

Human plasma fibronectin (pFN) contains a cryptic metalloprotease present in the collagen-binding domain. The enzyme could be generated and activated in the presence of Ca²⁺ from the purified 70-kDa pFN fragment produced by cathepsin D digestion. In this work we cloned and expressed the metalloprotease, designated FN type IV collagenase (FnColA), and a truncated variant (FnColB) in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, and subjected to folding and autocatalytic degradation in the presence of Ca²⁺, and yielded an active enzyme capable of digesting gelatin, helical type II and type IV collagen, - and -casein, insulin b-chain, and a synthetic Mca-peptide. In contrast, isolated plasma fibronectin, type I collagen, and the DNP-peptide were no substrates. Both catalytically active recombinant pFN fragments were efficiently inhibited by EDTA, and batimastat, and, in contrast to the glycosylated enzyme isolated from plasma fibronectin, were also inhibited by TIMP-2.     

Cell surface fibronectin of mouse peritoneal macrophages

Fibronectin (FN) was detected on thioglycollate-induced mouse peritoneal macrophages by binding the 125I-labeled F(ab')2 fragment of rabbit anti-human plasma fibronectin. The cell surface fibronectin (sFN) was removed from the surface of the macrophage monolayer by limited trypsinization. After trypsinization, binding of 125I-labeled plasma fibronectin (125I-pFN) to the macrophage monolayer was increased, suggesting that the FN receptor covered with sFN was exposed by trypsinization without destroying the receptor activity. The amounts of saturation binding of 125I-pFN to the macrophage monolayers before and after trypsinization were about 2.4 and 6.3 micrograms per 10(6) cells, respectively, indicating that the macrophage monolayer has the capacity of binding 6.3 micrograms FN per 10(6) cells, and the FN receptor equivalent to about 4 micrograms pFN per 10(6) cells is covered with sFN.     

Fibronectin-mediated binding and phagocytosis of polystyrene latex beads by baby hamster kidney cells

The binding and phagocytosis of fibronectin (pFN)-coated latex beads by baby hamster kidney (BHK) cells was studied as a function of fibronectin concentration and bead diameter. Cells were incubated with radioactive pFN-coated beads, and total bead binding (cell surface or ingested) was measured as total radioactivity associated with the cells. Of the bound beads, those that also were phagocytosed were distinguished by their insensitivity to release from the cells by trypsin treatment. In continuous incubations, binding of pFN-coated beads to cells occurred at 4 degrees C or 37 degrees C, but phagocytosis was observed only at 37 degrees C. In addition, degradation of 3H-pFN from ingested beads occurred at 37 degrees C, as shown by the release of trichloroacetic acid-soluble radioactivity into the incubation medium. When the fibronectin density on the beads was varied, binding at 4 degrees C and ingestion at 37 degrees C were found to have the same dose-response dependencies, which indicated that pFN densities that permitted bead binding were sufficient for phagocytosis to occur. The fibronectin density for maximal binding of ingestion was approximately 250 ng pFN/cm2. When various sized beads (0.085-1.091 micron), coated with similar densities of pFN, were incubated with cells at 4 degrees C, no variation in binding as a function of bead size was observed. Under these conditions, the absolute amount of pFN ranged from less than 100 molecules on the 0.085-micron beads to greater than 15,000 molecules on the 1.091-micron beads. Based upon these results it can be concluded that the critical parameter controlling fibronectin-mediated binding of latex beads by BHK cells is the spacing of the pFN molecules on the beads. Correspondingly, it can be suggested that the spacing between pFN receptors on the cell surface that is optimal for multivalent interactions to occur is approximately 18 nM. When phagocytosis of various sized beads was compared, it was found that the largest beads were phagocytosed slightly better (two fold) than the smallest beads. This occurred both in continuous incubations of cells with beads and when the beads were prebound to the cells. Finally, the kinetic constants for the binding of 0.085 microM pFN-coated beads to the cells were analyzed. There appeared to be approximately 62,000 binding sites and the KD was 4.03 X 10(-9) M. Assuming a bivalent interaction, it was calculated that BHK cells have approximately 120,000 pFN receptors/cell and the binding affinity between pFN and its receptor is approximately 6 X 10(-5) M.     

A major fraction of fibronectin present in the extracellular matrix of tissues is plasma-derived

The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA(+)FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA(+)FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues.     

Evidence for the recycling nature of the fibronectin receptor of macrophages

Plasma fibronectin (pFN) has been shown to mediate phagocytosis of several types of artificial particles and tissue debris by macrophages. In the present investigation some of the dynamic aspects of this receptor-mediated cellular process have been studied. Plasma fibronectin did not bind specifically to fibronectin (FN)-receptors of rat peritoneal macrophages at either 4 degrees C or 37 degrees C. On the other hand, pFN aggregated on the surface of gelatin-coated latex beads (gLtx) and 125I-labeled pFN covalently coupled to latex beads (pFN-Ltx) bound strongly to macrophages at both temperatures. Both of these particles were also internalized at 37 degrees C. Treatment of macrophages by chymotrypsin, thermolysin, or trypsin in a protein-free tissue culture medium did not affect either of the above reactions; however, pronase treatment strongly reduced both the binding and internalization of the pFN-coated particles. The pronase-treated macrophage monolayers in time regained their ability to bind and internalize pFN-gLtx when incubated in fresh tissue culture medium. Such recovery, however, did not take place when the medium contained cycloheximide. On the other hand, phagocytosis of pFN-gLtx was not affected directly by cycloheximide with untreated macrophages; this suggests that the FN-receptor recycles during sustained phagocytosis. This assumption was substantiated by the observations that some of the established lysosomotropic amines--i.e., chloroquine, dansylcadaverine, and dimethyldansylcadaverine--caused total inhibition of internalization without affecting the binding of particles to macrophages. Furthermore, chloroquine protected the FN-receptors against destruction by pronase. Together these results suggest that macrophage receptors for FN are protein, present both on the cell surface and intracellularly, and recycle between the plasma membrane and intracellular sites during phagocytosis.