Isolation and characterization of dermatan sulfate proteoglycans synthesized by cultured bovine aortic endothelial cells

Three glucuronic acid-rich dermatan sulfate proteoglycans (DS-PGs) have been isolated by chromatographic and electrophoretic techniques from cultures of bovine aortic endothelial cells and characterized structurally. The smallest of the DS-PGs (DS-II) has an apparent Mr of approximately 100,000 and glycosaminoglycan chains of Mr approximately 29,000. Core glycoprotein samples prepared by chondroitin ABC lyase digestion run as doublets of Mr = 45,000 and 48,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A decrease in core size is apparent after N-glycanase digestion, or when DS-PG is isolated from tunicamycin-treated cultures, providing evidence that the core protein is N-glycosylated. Isolated DS-II shows evidence of self-association when subjected to liquid chromatography under conditions of reduced ionic strength, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, DS-II, but not other endothelial cell DS-PG subclasses, is bound by an antibody against human skin fibroblast DS-PG, indicating that this DS-PG belongs to a family of widely distributed small DS-PGs, previously isolated from various connective tissues. A slightly larger (Mr approximately 220,000) DS-PG (DS-I) can be separated from DS-II by preparative electrophoresis. Despite similarities in core size and extent of N-glycosylation between DS-I and DS-II, DS-I shows only limited ability to self-associate, and does not interact with the anti-fibroblast DS-PG antibody. DS-I glycosaminoglycan chains are also smaller (Mr approximately 18,000) than those from DS-II, similar in size to the chains borne by the DS-PG subclass of largest size (high molecular weight (HMW)-DS). HMW-DS, which predominated in cell layer extracts, runs with a Kav of 0.45 on Sepharose CL-2B and is estimated to have an Mr greater than 700,000. Reduction and alkylation of HMW-DS indicates that it forms disulfide-bonded aggregates with other matrical proteins within the cell layer. HMW-DS displayed multiple protein cores (Mr greater than 200,000) upon chondroitin ABC lyase treatment. Despite some similarity in size to the family of large, aggregating chondroitin sulfate proteoglycans and DS-PGs, immunological evidence suggests that it lacks a hyaluronic acid binding region.     

Granzyme B cleavage of fibronectin disrupts endothelial cell adhesion,migration and capillary tube formation

Dysregulated angiogenesis contributes to the pathogenesis of chronic inflammatory diseases. Modulation of the extracellular matrix by immune-derived proteases can alter endothelial cell–matrix interactions as well as endothelial cell sprouting, migration and capillary formation. Granzyme B is a serine protease that is expressed by a variety of immune cells, and accumulates in the extracellular milieu in many chronic inflammatory disorders that are associated with dysregulated angiogenesis. Although granzyme B is known to cleave fibronectin, an essential glycoprotein in vascular morphogenesis, the role of granzyme B in modulating angiogenesis is unknown. In the present study, granzyme B cleaved both plasma fibronectin and cell-derived fibronectin, resulting in the release of multiple fibronectin fragments. Granzyme B cleavage of fibronectin resulted in a dose-dependent reduction in endothelial cell adhesion to fibronectin as well as reduced endothelial cell migration and tubular formation. These events were prevented when granzyme B activity was inhibited by a small molecule inhibitor. In summary, granzyme B-mediated cleavage of fibronectin contributes to attenuated angiogenesis through the disruption of endothelial cell — fibronectin interaction resulting in impaired endothelial cell migration and tubular formation.     

Interaction of small dermatan sulfate proteoglycan from fibroblasts with fibronectin

Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.     

Factor XIII cross-linking of fibronectin at cellular matrix assembly sites

We describe the effect of activated Factor XIII (Factor XIIIa, plasma transglutaminase) on the incorporation of plasma fibronectin into extracellular matrix by cultured human fibroblasts. In the absence of added Factor XIIIa, fibronectin binds to cultured fibroblast cell layers and is assembled into disulfide-bonded multimers of the extracellular matrix. When Factor XIIIa was included in the binding medium of skin fibroblasts, accumulation of 125I-fibronectin in the deoxycholate-insoluble matrix was increased. Fibronectin accumulating in the cell layer was cross-linked into nonreducible high molecular weight aggregates. The 70-kDa amino-terminal fragment of fibronectin inhibited the binding and cross-linking of 125I-fibronectin to cell layers, whereas fibrinogen had little effect. When 125I-fibronectin was incubated with isolated matrices or with cell layers pretreated with cytochalasin B, it did not bind and could not be cross-linked by Factor XIIIa into the matrix. HT-1080 human fibrosarcoma cells bound exogenous fibronectin following treatment with dexamethasone; Factor XIIIa cross-linked the bound fibronectin and caused its efficient transfer to the deoxycholate-insoluble matrix. These results indicate that exogenous fibronectin is susceptible to Factor XIIIa-catalyzed cross-linking at cellular sites of matrix assembly. Thus, Factor XIIIa-mediated fibronectin cross-linking complements disulfide-bonded multimer formation in the stabilization of assembling fibronectin molecules and thus enhances the formation of extracellular matrix.     

Retrovirally mediated expression of decorin by macrovascular endothelial cells. Effects on cellular migration and fibronectin fibrillogenesis in vitro

Decorin is a member of the widely expressed family of small leucine-rich proteoglycans. In addition to a primary role as a modulator of extracellular matrix protein fibrillogenesis, decorin can inhibit the cellular response to growth factors. Decorin expression is induced in endothelial cells during angiogenesis, but not when migration and proliferation are stimulated. Thus, decorin may support the formation of the fibrillar pericellular matrix that stabilizes the differentiated endothelial phenotype during the later stages of angiogenesis. Therefore, we tested whether constitutive decorin expression alone could modify endothelial cell migration and proliferation or affect pericellular matrix formation. To this end, replication-defective retroviral vectors were used to stably express bovine decorin, which was detected by Northern and Western blotting. The migration of endothelial cells that express decorin is significantly inhibited in both monolayer outgrowth and microchemotaxis chamber assays. The inhibition of cell migration by decorin was not accompanied by decreased proliferation. In addition, endothelial cells that express decorin assemble an extensive fibrillar fibronectin matrix more rapidly than control cells as assessed by immunocytochemical and fibronectin fibrillogenesis assays. These observations suggest that cell migration may be modulated by the influence of decorin on the assembly of the cell-associated extracellular matrix.     

Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix

Transforming growth factor beta (TGF-beta) is angiogenic in vivo. In vitro, endothelial cell proliferation is inhibited by TGF-beta. We have correlated this inhibitory effect with an increase in cellular fibronectin synthesis and deposition in a two-dimensional culture system using specific matrix coatings. The inhibitory effect was mimicked by addition of soluble fibronectin to cultures. In contrast, TGF-beta was found to elicit the formation of tube-like structures (mimicking angiogenesis) when microvascular endothelial cells were grown in three-dimensional collagen gels. In this culture system TGF-beta elicited rapid extensive formation of complex, branching, tube-like structures, while cell proliferation was not inhibited. These data confirm and support the hypothesis that TGF-beta is angiogenic and may exert some of its effects through modulation of matrix synthesis and are consistent with the hypothesis that the organization of the extracellular environment influences cellular responses to this "panregulin."     

Changes in proteoglycan composition during development of rat skin. The occurrence in fetal skin of a chondroitin sulfate proteoglycan with high turnover rate

Extraction of the skin of newborn rat yielded two populations of galactosaminoglycan-containing proteoglycan: a Mr = 111,000-200,000 dermatan sulfate proteoglycan (DS-PG) with a Mr congruent to 55,000 core glycoprotein and a Mr congruent to 10(6) chondroitin sulfate proteoglycan (CS-PGs) composed of two subpopulations with different size core-glycoproteins (Mr congruent to 480,000 and 520,000). Tryptic peptide mapping of chondroitinase-treated DS-PG and CS-PGs indicated that the peptide patterns observed with the two core molecules from CS-PGs were identical with each other but distinct from the peptide pattern of the DS-PG core molecule. It is likely therefore that the two forms of CS-PGs are derived from the same gene product by post-translational modification or partial degradation, but DS-PG is derived from a distinct gene product. Comparison of the concentration (hexuronate/DNA) of the proteoglycans in newborn and fetal rat skin showed an age-related change in proteoglycan composition; at 4 days before birth, the uronic acid proportions, DS-PG:CS-PGs, were about 14:1 and during the next 4 days, DS-PG increased 2.2-fold whereas CS-PGs decreased 4-fold. On a per DNA basis, the rate of [3H]serine incorporation into CS-PGs was 2.5 times the rate for DS-PG at 4 days before birth but decreased by 95% during the next 4 days. The rate for DS-PG also decreased but to a much lesser extent, so that by 2 days before birth, it began to exceed the rate for CS-PGs. The striking change in the concentration and labeling rate of CS-PGs can be interpreted either as a decrease of CS-PGs synthesis, or as an increase of CS-PGs breakdown, or both, a process which might be involved in the transition of extracellular matrix from a fetal type to a newborn or adult type.     

Factor XIIIa-mediated cross-linking of fibronectin in fibroblast cell layers. Cross-linking of cellular and plasma fibronectin and of amino-terminal fibronectin fragments

Factor XIIIa cross-links plasma fibronectin as it is being assembled into the extracellular matrix of cultured human skin fibroblasts (Barry, E. L. R., and Mosher, D. F. (1988) J. Biol. Chem. 262, 10464-10469). We have further characterized this process. Fibroblasts were metabolically labeled with proline in the presence or absence of ascorbate and Factor XIIIa. Endogenous fibronectin in the extracellular matrix was cross-linked by Factor XIIIa. There was no evidence for cross-linking of collagenous proteins. Fibro-blast cell layers were incubated with iodinated 27-kDa heparin-binding or 70-kDa collagen- and heparin-binding amino-terminal fibronectin fragments. Factor XIIa cross-linked the fragments into high molecular weight aggregates. The amounts of cross-linked fragments reaches a steady state after 1 to 2 h, whereas intact fibronectin continues to be cross-linked for 24 h. When fibroblast cell layers were pulsed with iodinated fibronectin or amino-terminal fragments and Factor XIIIa was included in the chase media, the high molecular weight aggregates were formed in a step-wise manner. The smallest cross-linking steps were to high molecular weight extracellular matrix molecules forming approximately 270-, 300-, and 440-kDa complexes for the 27-kDa fragment, 70-kDa fragment, and intact fibronectin, respectively. When iodinated fibronectin was bound to fibroblast cell layers and chased into the matrix pool in the absence of Factor XIIIa, it could also be cross-linked into high molecular weight complexes when Factor XIIIa was added to the media. These results, therefore, indicate that both cellular and plasma fibronectin and amino-terminal fragments are cross-linked specifically by Factor XIIIa, that the cross-linking is probably to other fibronectin molecules rather than to collagenous proteins, and that both assembling and assembled fibronectin are substrates for Factor XIIIa.     

Peptides derived from human decorin leucine-rich repeat 5 inhibit angiogenesis

Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 3(10) helix and beta-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs.     

Ganglioside-dependent adhesion events of human neuroblastoma cells regulated by the RGDS-dependent fibronectin receptor and proteoglycans

Human neuroblastoma cells (Platt and La-N1) adhere and extend neurites on a ganglioside GM1-binding substratum provided by cholera toxin B (CTB). These adhesive responses, similar to those on plasma fibronectin (pFN), require the mediation of one or more cell-surface proteins [G. Mugnai and L. A. Culp (1987) Exp. Cell Res. 169, 328]. The involvement of two pFN receptor molecules in ganglioside GM1-mediated responses on CTB have now been tested. In order to test the role of cellular FN binding to its glycoprotein receptor integrin, a soluble peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence was added to the medium. It did not inhibit attachment on CTB but completely inhibited formation of neurites; in contrast, the RGDS peptide minimally inhibited attachment or neurite formation on pFN. Once formed, neurites on CTB became resistant to the peptide. In order to test the role of cell-surface heparan sulfate proteoglycan (HS-PG), two approaches were used. First, the HS-binding protein platelet factor-4 (PF4) was used to dilute CTB or pFN on the substratum or, alternatively, added to the medium. Diluting the substratum ligand with PF4 had no effects on attachment on either CTB or pFN. However, neurite formation on CTB was readily inhibited and on pFN partially inhibited; the effects of PF4 were far greater than a similar dilution with nonbinding albumin. When PF4 was added to the medium of cells, attachment on either substratum was unaffected as was neurite outgrowth on pFN, revealing differences in PF4's inhibition as the substratum-bound or medium-borne component. In contrast, PF4 in the medium at low concentrations (1 microgram/ml) was highly inhibitory for neurite formation on CTB. The second approach utilized the addition of bovine cartilage dermatan sulfate proteoglycan (DS-PG), shown to bind to pFN as well as to substratum-bound CTB by ELISA, or cartilage chondroitin sulfate/keratan sulfate proteoglycan (CS/KS-PG) to the substratum or to the medium. At low concentrations, DS-PG but not CS/KS-PG actually stimulated neurite formation on CTB while at higher concentrations DS-PG completely inhibited attachment and neurite formation. While DS-PG partially inhibited attachment on pFN, it had no effect on neurite formation of the attached cells. Neuroblastoma cells adhered to some extent to substrata coated only with DS-PG, indicating "receptors" for PGs that permit stable interaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transglutaminase 2 is secreted from smooth muscle cells by transamidation-dependent microparticle formation

Transglutaminase 2 (TG2) is a pleiotropic enzyme involved in both intra- and extracellular processes. In the extracellular matrix, TG2 stabilizes the matrix by both covalent cross-linking and disulfide isomerase activity. These functions become especially apparent during matrix remodeling as seen in wound healing, tumor development and vascular remodeling. However, TG2 lacks the signal sequence for a classical secretory mechanism, and the cellular mechanism of TG2 secretion is currently unknown. We developed a green fluorescent TG2 fusion protein to study the hypothesis that TG2 is secreted via microparticles. Characterization of TG2/eGFP, using HEK/293T cells with a low endogenous TG2 expression, showed that cross-linking activity and fibronectin binding were unaffected. Transfection of TG2/eGFP into smooth muscle cells resulted in the formation of microparticles (MPs) enriched in TG2, as detected both by immunofluorescent microscopy and flow cytometry. The fraction of TG2-positive MPs was significantly lower for cross-linking-deficient mutants of TG2, implicating a functional role for TG2 in the formation of MPs. In conclusion, the current data suggest that TG2 is secreted from the cell via microparticles through a process regulated by TG2 cross-linking.     

Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking

We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups.     

Monoclonal antibody DH12 reacts with a cell surface and a precursor form of the beta subunit of the human fibronectin receptor

Monoclonal and polyclonal antibodies were raised against a placenta plasma membrane protein preparation, which was obtained by fractionation on Blue B dye matrix and by HPLC-anionexchange, and which was shown to contain fibronectin receptors. Immunochemical and functional evidence showed that monoclonal antibody DH12 recognized the beta subunit of the human fibronectin receptor on fibroblasts. This monoclonal antibody reacted with two proteins in Western blots and in double immune precipitations of whole cell preparations. Only the higher Mr protein became labeled by surface iodination of intact fibroblasts. The lower Mr protein is thought to be an intracellular precursor of the beta subunit of the fibronectin receptor.     

Tenascin-C inhibits beta1 integrin-dependent cell adhesion and neurite outgrowth on fibronectin by a disialoganglioside-mediated signaling mechanism

We have previously shown that the extracellular matrix molecule tenascin-C inhibits fibronectin-mediated cell adhesion and neurite outgrowth by an interaction with a cellular RGD-independent receptor which interferes with the adhesion and neurite outgrowth promoting activities of the fibronectin receptor(s). Here we demonstrate that the inhibitory effect of tenascin-C on beta1integrin-dependent cell adhesion and neurite outgrowth is mediated by the interaction of the protein with membrane-associated disialogangliosides, which interferes with protein kinase C-related signaling pathways. First, in substratum mixtures with fibronectin, an RGD sequence-containing fragment of the molecule or synthetic peptide, tenascin-C inhibited cell adhesion and spreading by a disialoganglioside-dependent, sialidase-sensitive mechanism leading to an inhibition of protein kinase C. Second, the interaction of intact or trypsinized, i.e., cell surface glycoprotein- free, cells with immobilized tenascin-C was strongly inhibited by gangliosides or antibodies to gangliosides and tenascin-C. Third, preincubation of immobilized tenascin-C with soluble disialogangliosides resulted in a delayed cell detachment as a function of time. Similar to tenascin-C, immobilized antibody to GD2 (3F8) or sphingosine, a protein kinase C inhibitor, strongly inhibited RGD- dependent cell spreading. Finally, the degree of tenascin-C-induced inhibition of cell adhesion was proportional to the degree of disialoganglioside levels of expression by different cells suggesting the relevance of such mechanism in modulating integrin-mediated cell- matrix interactions during pattern formation or tumor progression.      

Identification of a fibronectin receptor specific for rat liver endothelial cells

Antibodies raised against the fibronectin receptor of rat hepatocytes recognized one protein (Mr 120 and 135 kDa for unreduced and reduced samples, respectively) in immunoblotting of solubilized rat liver endothelial cells (LEC). The antibodies specifically precipitated a 200-kDa protein together with the 135-kDa component from 125I-labeled LEC. Spreading of LEC on fibronectin, but not on laminin or collagen, was inhibited by monovalent Fab fragments of the antibodies, implicating that the 135/200-kDa complex is a specific fibronectin receptor. The results indicate that LEC, hepatocytes, and fibroblasts of rat carry different fibronectin receptors, suggesting that the interaction of fibronectin with these cells may have different functional roles.     

Identification of integrin-like matrix receptors with affinity for interstitial collagens

Antibodies to a rat liver membrane glycoprotein with an Mr of 115,000 (nonreduced) inhibited the attachment of rat hepatocytes and primary rat heart fibroblasts to both collagen and fibronectin. The Mr 115,000 glycoprotein cross-reacted immunologically with the beta 1-chain of the rat hepatocyte fibronectin receptor (HFNR), and the two proteins showed identical peptide maps after proteolytic cleavage. It was concluded that the Mr 115,000 protein was similar or identical to the beta 1-chain of Arg-Gly-Asp (RGD)-directed matrix receptors. Although collagen type I contains several RGD sequences, the attachment of hepatocytes and fibroblasts to collagen type I was not inhibited by the synthetic peptide GRGDTP in concentrations that blocked adhesion to fibronectin. Furthermore, hepatocytes adhered equally well to collagen fragments, generated by cyanogen bromide cleavage, lacking RGD sequences as to fragments containing this sequence. Antibodies to the Mr 115,000 protein inhibited the adhesion of hepatocytes to both types of collagen fragments. Taken together, these data indicate the presence of collagen receptors that share the beta-subunit with the HFNR but that are not directed to RGD sequences. Tentative alpha-chains of the collagen matrix receptor complex were isolated by immunoprecipitation of surface 125I-labeled fibroblast membrane proteins purified by affinity chromatography on immobilized collagen type I. Data are presented indicating that proteins with Mr around 145,000 and 170,000 (nonreduced) are associated in noncovalently linked complexes with the Mr 115,000 protein. These complexes have affinity for collagen and thus have properties expected for integrin-like collagen receptors.     

The elastin associated glycoprotein gp115. Synthesis and secretion by chick cells in culture

Synthesis of gp115 by aorta smooth muscle cells and tendon fibroblasts isolated from chick embryos was investigated. gp115 was specifically immunoprecipitated by both polyclonal and monoclonal antibodies from cell lysates and culture medium of matrix free cells metabolically labeled with [3H]leucine and [35S]methionine. The component of gp115 isolated from the cell lysate had an apparent Mr in reduced sodium dodecyl sulfate polyacrylamide gels lower (105,000) than the protein isolated from the culture medium (Mr = 115,000). In immunoblot experiments, the latter corresponded in apparent Mr to the form isolated from chick tissues. gp115 was glycosylated in vitro; it was labeled with [3H]fucose, and when cells were cultured and labeled in the presence of tunicamycin, a lower Mr form with an apparent Mr = 90,000 was immunoprecipitated in both the cell lysate and the culture medium. In pulse-chase experiments, the intracellular and the extracellular forms were clearly suggestive of a direct precursor-product relationship in the absence of intermediate forms. The kinetics of secretion appeared very slow compared with that of other proteins of the extracellular matrix investigated in the same system; about 50-70% of gp115 in the form of the Mr = 105,000 species was still cell-associated after 4 h, whereas the half-time for secretion of fibronectin, type VI collagen, and tropoelastin was about 60 min, 3 h, and 60 min, respectively. Newly synthesized and processed cell-associated gp115 migrated in both reduced and non-reduced gels as a monomer. On the contrary, the secreted protein was present in the culture medium as large aggregates that did not enter the gel in the absence of reducing agents.     

Collagen type I together with fibronectin provide a better support for endothelialization

Endothelialization of vascular implants is limited by the inability of cells to retain adhesion when exposed to flow. Extracellular matrix proteins, including fibronectin and collagen, enhance cell adherence on materials. This study investigated the behaviour of Human Umbilical Vein Endothelial Cells (HUVEC) on extracellular matrix coated polystyrene. Collagen and fibronectin were coated as single and double layers to analyse differences in cell proliferation, morphology, and cell-protein interactions. Significantly higher endothelial cell proliferation and migration rates were observed on the collagen and collagen+fibronectin coating compared to the uncoated or fibronectin-coated sample. Immmunofluorescent microscopy showed evidence of extracellular matrix remodelling in the double, collagen+fibronectin coating. These results strongly suggest that a double coating of collagen+fibronectin provides a better support structure for endothelial cell growth and contributes to improve the ability of vascular implants to become and remain endothelialized.     

Effects of fibronectin on hydroxyapatite formation.

There is increasing evidence that noncollagenous matrix proteins initiate bone mineralization in vivo. Fibronectin, which is present during the early phases of mineralization, may contribute to this process in bone tissues. In this context, the mineralization potential of fibronectin was tested in an agarose gel precipitation system and a metastable calcium phosphate solution. The protein inhibited the precipitation of calcium phosphate crystals in solution but had no apparent effect in gel. Conversely, fibronectin stimulated crystal formation when apatite powder was used to seed crystal growth in gel. Although these results in vitro do not clearly indicate that fibronectin is involved in the mineralization process, they are consistent with in vivo events. Free fibronectin (e.g. in biological fluids) could inhibit crystal growth but might also activate the mineralization process when absorbed on apatite powder in a bone environment and areas of ectopic mineralization.     

The Laminin–Nidogen Complex is a Ligand for a Specific Splice Isoform of the Transmembrane Protein Tyrosine Phosphatase LAR

Leukocyte antigen–related protein (LAR) is a prototype for a family of transmembrane protein tyrosine phosphatases whose extracellular domain is composed of three Ig and several fibronectin type III (FnIII) domains. Complex alternative splicing of the LAR-FnIII domains 4–8 has been observed. The extracellular matrix laminin–nidogen complex was identified as a ligand for the LAR-FnIII domain 5 (Fn5) using a series of GST-LAR-FnIII domain fusion proteins and testing them in in vitro ligand-binding assays. LAR– laminin–nidogen binding was regulated by alternative splicing of a small exon within the LAR-Fn5 so that inclusion of this exon sequence resulted in disruption of the laminin–nidogen-binding activity. Long cellular processes were observed when HeLa cells were plated on laminin–nidogen, but not when plated on a fibronectin surface. Indirect immunofluorescent antibody staining revealed high expression of LAR in a punctate pattern, throughout the length of these cellular processes observed on laminin–nidogen. Antibody-induced cross-linking of LAR inhibited formation of these cellular processes, and inhibition was correlated with changes in cellular actin cytoskeletal structure. Thus, LAR–laminin–nidogen binding may play a role in regulating cell signaling induced by laminin–nidogen, resulting in cell morphological changes.