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EA.hy 926 cells, a derivative of human umbilical vein endothelial cells, in the presence of fibroblasts show the phenomena of angiogenesis, express the proteoglycan decorin and escape apoptosis, when they are maintained in collagen lattices, while fibroblast-free cultures do not show these changes. Virus-mediated decorin expression can substitute for the presence of fibroblasts. Since the expression of matrix metalloproteinases (MMPs) is an essential step in the formation of capillaries, several MMPs and tissue inhibitors of metalloproteinases (TIMPs) were investigated. MMP-1, MMP-2, MMP-9, and the cell-associated MMP-14 were augmented on the protein level in the presence of fibroblasts. No effect was seen with respect to MMP-3, TIMP-1, and TIMP-2. Semiquantitative RT-PCRs of endothelial cells in co-culture revealed a 7-, 19-, and 11-fold increase for mRNAs of MMP-1, MMP-2, and MMP-14 after six days, respectively. Virus-mediated decorin expression also was accompanied by an up-regulation of these MMPs. The expression of MMP-1 mRNAs increased 5-fold after 2 days and gradually declined thereafter. In contrast, MMP-2 and MMP-14 showed a 7-fold and a 14-fold increase on day two which returned to basal levels within 24 h, indicating that the expression of MMP-1 is differentially regulated from MMP-2 and MMP-14. In spite of the upregulation of the proteases, an enhanced degradation of decorin was not observed. These results indicate that the expression of decorin is a sufficient signal in EA.hy 926 cells for a finely tuned induction of selected MMPs which are involved in angiogenesis whereas the up-regulation of MMPs does not lead to the degradation of the responsible proteoglycan., (Copyright 2001 Wiley-Liss, Inc.)

Complex formation of transforming growth factor-beta (TGF-beta) with the small proteoglycan decorin has been considered to inactivate the cytokine. However, neither the TGF-beta-mediated stimulation of the retraction of collagen lattices in culture nor the enhanced transcription of biglycan were influenced by an excess of native decorin in the culture medium. In contrast, when MG-63 osteosarcoma cells were transfected with sense- or antisense-decorin-cDNA, which led to an over- or under-expression of the proteoglycan, they responded to TGF-beta differently. An inverse correlation between decorin expression and the TGF-beta-mediated stimulation of collagen gel retraction and biglycan induction, respectively, was found. These results are best explained by assuming that decorin is not inactivating but sequestering TGF-beta in the extracellular matrix.

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.

It has been shown that small proteoglycans containing leucine-rich repeats in their core proteins can form complexes with TGF-beta. Decorin, a ubiquitously found molecule of the extracellular matrix, is the best-studied example. Therefore, binding domains on its core protein were investigated using recombinant decorin fragments generated as fusion proteins in prokaryotes. The peptide Leu155-Val260 immobilized by the polyhistidine tag on a nickel chelate column bound TGF-beta1 and -beta2 almost as effectively as the largest fragment (Asp45-Lys359) studied. Other peptides were less effective. For the two peptides Asp45-Lys359 and Leu155-Val260 dissociation constants in the nanomolar range for high-affinity binding sites were calculated in a solid-phase assay with immobilized TGF-beta2. Peptide Asp45-Lys359 also contained a lower affinity binding site. Domains with lower affinity were also found in peptides Asp45-Leu155 and Arg63-Gly190. Peptide Leu155-Val260 also formed complexes with TGF-beta in the liquid phase as determined by equilibrium gel filtration. Furthermore, F(ab') fragments of polyclonal antibodies against peptide Leu155-Val260 interfered with TGF-beta binding to peptide Asp45-Lys359 in a dose-dependent manner. Peptide Leu155-Val260, however, is only a weak competitor of the binding of wild-type decorin to reconstituted type I collagen fibrils. Therefore, independent binding sites of decorin for TGF-beta and type I collagen should exist. In support of this hypothesis saturable binding of TGF-beta1 and TGF-beta2 to collagen-bound native decorin could be demonstrated. The bound cytokine could be released in a biologically active form by collagenase treatment. Thus, decorin may play a biological role in storing this cytokine temporarily in the extracellular matrix and in thereby modulating an interaction of TGF-beta with its signaling receptors., (Copyright 1998 Academic Press.)

Through its ability to bind extracellular matrix constituents and growth factors the small leucine-rich chondroitin/dermatan sulfate proteoglycan decorin which is present in many types of connective tissues may play an important biological role in remodeling and maintenance of extracellular matrices during inflammation, fibrosis, and cancer growth. In this study we investigated the known binding of decorin to human collagen XIV. This binding was unaffected when the small collagenous moiety of collagen XIV was removed with collagenase. Therefore, fragments covering the large noncollagenous domain NC3 of collagen XIV were expressed in Escherichia coli, each fused to a 26-kDa fragment of glutathione S-transferase. Using radioiodinated decorin as ligand for the immobilized fusion proteins, a binding site that interacted with the decorin core protein could be assigned to the NH2-terminal fibronectin type III repeat of collagen XIV. In addition, an auxiliary binding site located COOH-terminal to this fibronectin type III repeat interacted with the glycosaminoglycan component of decorin.

The chondroitin/dermatan sulfate proteoglycan decorin is known to interact via its core protein with fibrillar collagens, thereby influencing the kinetics of fibril formation and the final diameter of the fibrils. To define the binding site(s) for type I collagen along the core protein, which is mainly composed of leucine-rich repeat structures, decorin cDNAs were constructed and expressed in human kidney 293 cells. The constructs encoded (i) C-terminally truncated molecules, (ii) core proteins with deletions of selected leucine-rich repeats, or (iii) various point mutations. The deletion of the sixth leucine-rich repeat Met176-Lys201 and the mutation E180K drastically interfered with the binding to reconstituted type I collagen fibrils. In contrast, the deletion of the seventh repeat Leu202-Ser222 led at the most to a marginally impaired binding, although the secretion of this proteoglycan was abnormally low. Decorin with two other point mutations in the sixth leucine-rich repeat, Lys187 --> Gln and Lys200 --> Gln, respectively, bound type I collagen either normally or even better than the normal recombinant proteoglycan. These data suggest that a major collagen-binding site of decorin is located within the sixth leucine-rich repeat and that glutamate-180 within this repeat is of special importance for ionic interactions between the two matrix components.

The small dermatan sulphate protein decorin interacts via its core protein with fibrillar collagens, and its glycosaminoglycan chains were proposed to be capable of self-association. It was therefore of interest to study the role of decorin in the contraction of cell-populated collagen lattices. Stable transfection of dihydrofolate reductase-deficient CHO cells with decorin cDNA resulted in impaired collagen lattice contraction. Using normal human skin fibroblasts in serum-free cultures, inclusion of 0.3 microM decorin in the culture medium also led to a delayed collagen gel contraction. Protein-free dermatan sulphate and the dermatan sulphate-degrading enzyme chondroitin ABC lyase were ineffective. Potential interactions between dermatan sulphate chains were studied by gel filtration. A shift in the elution position of [35S]sulphate-labelled decorin-derived glycosaminoglycans by unlabelled decorin could be observed only when the chains were prepared by trypsin. Chains liberated by beta-elimination or by cathepsin C were eluted at identical positions in the presence or absence of decorin. It is therefore unlikely, that the effect of decorin on collagen-gel retraction is brought about solely by glycosaminoglycan-glycosaminoglycan interactions.

Interactions between the core protein of the small dermatan sulfate proteoglycan decorin and type I collagen have been considered to influence the kinetics of collagen fibrillogenesis and the diameter of and the distance between the fibrils. A variety of recombinant core protein fragments were expressed in Escherichia coli, extracted from inclusion bodies, and renatured in the presence of bovine serum albumin, which was essential for obtaining functional activity. A recombinant protein lacking the first 14 amino acids of the mature core protein (P15-329) interacted with reconstituted type I collagen fibrils and inhibited collagen fibrillogenesis almost as efficiently as intact decorin purified from fibroblast secretions under non-denaturing conditions. Peptides comprising amino acids 15-183 (P15-183) and 185-329 (P185-329) were able to compete for the binding of wild-type decorin, with P15-183 being more active than P185-329. Several other peptides were much less effective. Binding studies using radioactively labeled peptides P15-183 and P185-329 gave direct evidence for the independent binding of both peptides. Peptides 15-183 and 15-125 had the capability of inhibiting collagen fibrillogenesis, whereas peptide 185-329 was inactive. These data suggest (i) that there are at least two separate binding domains for the interaction between decorin core protein and type I collagen and (ii) that binding is not necessarily correlated with an alteration of collagen fibrillogenesis.

The small proteoglycan decorin is known to interact with type I collagen fibrils, thereby influencing the kinetics of fibril formation and the distance between adjacent collagen fibrils. The structurally related proteoglycan biglycan has been proposed not to bind to fibrillar collagens. However, when osteosarcoma cells were cultured on reconstituted type I collagen fibrils, both decorin and biglycan were retained by the matrix. Immunogold labeling at the electron microscopic level showed that both proteoglycans were distributed along collagen fibrils not only in osteosarcoma cell-populated collagen lattices but also in human skin. Reconstituted type I collagen fibrils were able to bind in vitro native and N-glycan-free biglycan as well as recombinant biglycan core protein. From Scatchard plots dissociation, constants were obtained that were higher for glycanated biglycan (8.7 x 10(-8) mol/liter) than for glycanated decorin (7 x 10(-10) mol/liter and 3 x 10(-9) mol/liter, respectively). A similar number of binding sites for either proteoglycan was calculated. Recombinant biglycan and decorin were characterized by lower dissociation constants compared with the glycanated forms. Glycanated as well as recombinant decorin competed with glycanated biglycan for collagen binding, suggesting that identical or adjacent binding sites on the fibril are used by both proteoglycans. These data suggest that, because of its trivalency, biglycan could have a special organizing function on the assembly of the extracellular matrix.

Small dermatan sulphate proteoglycan II from cultured human skin fibroblasts interacts with type I collagen in vitro and in vivo. When fibroblasts are maintained in a type I collagen lattice the proteoglycan remains exclusively within the lattice, and its association with fibrils can be demonstrated immunocytochemically. On the basis of [35S]sulphate incorporation, small proteoglycan II comprises about 80% of total proteoglycans secreted by cells in monolayer culture. In a collagen lattice, fibroblasts down-regulate its synthesis to the level of large chondroitin sulphate/dermatan sulphate and of heparan sulphate proteoglycans, the synthesis of which remains unaffected. Compared with the product from monolayer cultures, small proteoglycan II from collagen gels contained a longer polysaccharide chain which is characterized by a larger proportion of disulphated and a smaller proportion of monosulphated glucuronic acid-containing disaccharides. The half-life varied between 60 and 110 h. It is suggested that the compositional differences between the proteoglycan from monolayer cultures and from cells in a collagen lattice are related to the slower intracellular trafficking of the proteoglycan under the latter culture conditions.

Human skin fibroblasts were cultivated within the three-dimensional space of polymerized alginate and collagen, respectively. The in vitro synthesis of collagens and proteoglycans was measured during the first 3 days of culture, and the deposition as well as the ultrastructural organization of newly synthesized extracellular matrix components were examined by electron microscopy. The amount of collagens and proteoglycans synthesized by fibroblasts, embedded in calcium alginate gels as well as in collagen lattices, was lowered as compared to monolayer cultures. Furthermore, it was found that collagen synthesis was reduced to a greater extent in alginate gels than in collagen lattices. On the contrary, total proteoglycan biosynthesis was similarly reduced either in alginate gels or in collagen lattices. At the end of a 3-day-culture period, filamentous material as well as cross-striated banded structures were found extracellularly in the alginate gel. According to their periodicity, their banding pattern, their association with polyanionic matrix components and their sensitivity towards glycosaminoglycan-degrading enzymes we could distinguish (1) sheets of amorphous non-banded material consisting of irregularly arranged filaments and containing dermatan sulfate-rich proteoglycans (type I structures), (2) sheets of long-spacing fibrils consisting of parallel orientated filaments and containing chondroitin sulfate-rich proteoglycans (= zebra bodies; type II structures), and (3) fibrillar structures with a complex banding pattern different from that of native collagen fibrils (type III structures). In fibroblasts cultured in collagen lattices, we only sporadically found depositions which are identified as type I structures. Using indirect immunoelectron microscopy and monospecific polyclonal antibodies, we localized type VI collagen in type I structures and type II structures. Type III structures can be identified as type I collagen derived as becomes obvious by comparison with segment long spacing crystallites of type I collagen.

Background: The small chondroitin/dermatan sulphate proteoglycans decorin and biglycan participate in organizing the network of collagen fibrils and interact with non-collagenous matrix proteins. In addition, via interactions with cytokines they are directly or indirectly involved in signalling, growth and cell differentiation. We aimed to analyse their expression in normal gastric tissue and during gastric ulcer healing. Methods: Proteoglycan expression was studied by immunohistochemistry and in situ hybridization in acetic acid-induced gastric ulcers in rat during early phases and during chronic ulceration. The effects of treatment with an acid stable mutein of FGF-2 (bFGF) were also studied. Results: In normal gastric tissue, both proteoglycans were most strongly expressed in the submucosal layer. However, some epithelial cells were positive for biglycan and, surprisingly, also for decorin. In the early phase after ulcer induction exclusively decorin became induced in the muscularis mucosae, while biglycan became detectable in this layer only after 2 weeks. There was no up-regulation of either proteoglycan in other layers, nor could an effect of FGF-2 treatment be seen. Conclusions: The expression of decorin could be observed for the first time in epithelial cells. Decorin, but not biglycan, appears as an early phase reactant in the muscularis mucosae in accordance with its putative role during angiogenesis and the prevention of apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Sessile organisms may experience chronic exposure to copper that is released into the marine environment from antifoulants and stormwater runoff. We have identified the site of damage caused by copper to the symbiotic cnidarian, Zoanthus robustus (Anthozoa, Hexacorallia). External changes to the zoanthids were apparent when compared with controls. The normally flexible bodies contracted and became rigid. Histological examination of the zoanthid tissue revealed that copper had caused sub-cellular changes to proteins within the extracellular matrix (ECM) of the tubular body. Collagen in the ECM and the internal septa increased in thickness to five and seven times that of controls respectively. The epithelium, which stained for elastin, was also twice as thick and tough to cut, but exposure to copper did not change the total amount of desmosine which is found only in elastin. We conclude that copper stimulated collagen synthesis in the ECM and also caused cross-linking of existing proteins. However, there was no expulsion of the symbiotic algae (Symbiodinium sp.) and no effect on algal pigments or respiration (44, 66 and 110µg Cu L− 1). A decrease in net photosynthesis was observed only at the highest copper concentration (156µg Cu L− 1). These results show that cnidarians may be more susceptible to damage by copper than their symbiotic algae. [Copyright &y& Elsevier]