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Extracellular matrix of lymphoid tissues in the chick.

We describe the immunohistochemical distribution of components of the extracellular matrix of the chick lymphoid system. In the thymus, basement membranes of epithelial cells bordering the lobules were intensely stained by laminin antibodies; fibronectin antibodies labeled the capsule and the septal matrix, and similar reactivity was seen with tropoelastin and gp 115 antibodies. No positivity was detected with any of the antibodies within the cortical parenchymal cells. Laminin was not detected in the medullary parenchyma, whereas fibronectin was present as coarse fibers. Tropoelastin and gp 115 appeared as a finer and more diffuse meshwork. In the bursa, laminin antibodies outlined the epithelial cells separating the cortex from the medulla. Fibronectin, tropoelastin, and gp 115 antibody stained the interfollicular septa and the cortical matrix, although to a different extent. Laminin was also detected in association with the interfollicular epithelium (IFE) basement membrane, whereas no staining was found underneath the follicle-associated epithelium (FAE). FAE cells not only lack a proper basement membrane but are also not separated from medullary lymphocytes by any of the other extracellular matrix components were investigated. Consequently, medullary lymphocytes are not sequestered, and can come easily into contact with antigens present in the intestinal lumen. All four antibodies stained the spleen capsule and spleen blood vessels, tropoelastin and gp 115 antibodies giving the strongest reactivity. A fine trabecular staining pattern was detected with gp 115 antibodies in the white pulp.

Animals↗

Domains of apolipoprotein E involved in the binding to the protein core of biglycan of the vascular extracellular matrix: potential relationship between retention and anti-atherogenic properties of this apolipoprotein.

We recently identified elements in the C-terminal domain of apolipoprotein E that are critical for the binding of this protein to the protein core of biglycan, a proteoglycan of the vascular extracellular matrix. This binding, ionic in nature, requiring no participation by glycosaminoglycans, suggests that protein-protein interactions may play an important role in the binding of apolipoprotein E to the extracellular matrix. These interactions may represent a potential mechanism for the anti-atherogenic potential of this apolipoprotein by preventing further anchoring to the vascular matrix of pro-atherogenic factors.

Apolipoproteins E↗

Extracellular matrix: an immunological and biochemical (CAT and TOH activity) survey of in vitro differentiation of isolated amphibian neuroblasts.

After neural induction certain cells in the neuroepithelium immediately acquire the property to express certain neural phenotypes (Duprat et al., 1984, 1987). However, the activity of almost all the specific enzymes involved in the biosynthesis of neurotransmitters is considerably higher when neurectodermal cells are cultured with chordamesodermal cells than when they are cultured alone. The stimulating effects of chordamesoderm do not appear to be due to diffusible factors (Duprat et al., 1985b). The present study was designed to investigate the role of extracellular matrix components in neuronal cell differentiation. We showed that the extracellular matrix cannot replace chordamesoderm in stimulating the biochemical differentiation of neuroblasts, although fibronectin and especially laminin stimulate morphological differentiation. We suggest that interaction between neuronal and non-neuronal cells plays an important part in functional biochemical differentiation, whereas the molecules of extracellular matrix are important for morphological differentiation.

Animals↗

Modeling the cholesteatoma microenvironment: coculture of HaCaT keratinocytes with WS1 fibroblasts induces MMP-2 activation, invasive phenotype, and proteolysis of the extracellular matrix.

BACKGROUND: Increased keratinocyte proliferation, increased keratinocyte migration, elaboration of proteases resulting in proteolysis of the extracellular matrix (ECM), and destruction of surrounding tissues all typify the course of cholesteatoma growth. The contribution of stromal fibroblasts to these behaviors remains relatively unexplored. OBJECTIVES: Our objective for the current studies was to create a simple model with which to study these cholesteatoma behaviors, specifically, cell migration, invasion, and proteolysis of the extracellular matrix as well as the role of fibroblasts in the activated keratinocyte phenotype of cholesteatoma. DESIGN: The authors conducted an in vitro culture model. METHODS: The resulting model consists of activated keratinocytes (HaCaT cells) cocultured with normal dermal fibroblasts (WS1 cells) within a three-dimensional reconstituted ECM. We used a confocal imaging assay and software analysis to quantify total functional proteolysis of the ECM in monotypic and organotypic cocultures. This was accomplished by growing cells on an artificial ECM comprised of Matrigel and DQ-collagen IV. DQ-collagen is a "quenched" fluorescent peptide whose fluorescence is unmasked by proteolytic cleavage. RESULTS: Organotypic cocultures of keratinocytes and fibroblasts exhibited increased cell migration, increased cell invasion, increased matrix metalloproteinase-2 secretion and activation, and increased proteolysis of type IV collagen in three-dimensional ECM. Exposure to NSC27366, inhibitor of the small GTPase, Rac, resulted in reduction in both cell invasion and ECM proteolysis. CONCLUSIONS: Stromal fibroblasts may stimulate the invasive phenotype of keratinocytes, including ECM proteolysis. Increased cell invasion and proteolysis are dependent on the Rac pathway in this model. This simple culture model may help further our understanding of these destructive behaviors in cholesteatoma keratinocytes.

Cell Communication↗

Reciprocal interactions between cells and extracellular matrix during remodeling of tissue constructs.

Cells remodel extracellular matrix during tissue development and wound healing. Similar processes occur when cells compress and stiffen collagen gels. An important task for cell biologists, biophysicists, and tissue engineers is to guide these remodeling processes to produce tissue constructs that mimic the structure and mechanical properties of natural tissues. This requires an understanding of the mechanisms by which this remodeling occurs. Quantitative measurements of the contractile force developed by cells and the extent of compression and stiffening of the matrix describe the results of the remodeling processes. Not only do forces exerted by cells influence the structure of the matrix but also external forces exerted on the matrix can modulate the structure and orientation of the cells. The mechanisms of these processes remain largely unknown, but recent studies of the regulation of myosin-dependent contractile force and of cell protrusion driven by actin polymerization provide clues about the regulation of cellular functions during remodeling.

Animals↗

Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) cleaves and releases a 22-kDa extracellular matrix metalloproteinase inducer (EMMPRIN) fragment from tumor cells.

Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.

Amino Acid Sequence↗

Modulation by hypergravity of extracellular matrix macromolecules in in vitro human dermal fibroblasts.

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity (20 g) over a period of 8 days. Changes in organization of extracellular matrix molecules were seen by indirect immunofluorescence. In the fibronectin layer, bundles of fibrils were gathered together leading to a disorganisation of the normal parallel pattern of fibers seen in control cultures. Type I collagen fibrils appeared with wooly outlines in controls whereas thick fibers were closely packed in 20-g cultures. A moderate increase of type III collagen fibril density was observed. No elastic fibers were seen in control or in 20-g cultures. In the culture medium, the release of soluble elastin (ELISA) and type I and III collagens (RIA) was undisturbed. Assays of enzymes involved in the remodeling of extracellular matrix showed an increase of cellular elastase activity (10%) and a decrease of the spontaneously active collagenase. Nevertheless, the total collagenase activity, (activated by trypsin), was increased by up to 30%. These data show a significant rise of the latent collagenase activity and suggest that release of the tissue inhibitor of metalloproteinase (TIMP1) was enhanced by hypergravity.

Adolescent↗

Characterization of cellular density and determination of neointimal extracellular matrix constituents in human lower extremity vein graft stenoses.

UNLABELLED: Arterial restenosis has been attributed to a hyperproliferative smooth muscle cell response. Paradoxically, studies of human coronary atherectomy and vein graft stenotic lesions have demonstrated a relatively low nuclear proliferative rate with the majority of the neointimal mass consisting of extracellular matrix. The purpose of the present study was to characterize the cellular density and determine the relative composition of the extracellular matrix protein constituents in stenotic, human lower extremity vein-bypass graft lesions. METHODS: Duplex surveillance of 148 consecutive infrainguinal bypass grafts identified 17 patients with 22 preocclusive autogenous vein graft stenoses (mean graft age 7 months). Morphological analyses of these stenotic lesions were compared with excised samples of 20 greater saphenous vein segments taken at the time of graft implantation from matched control patients. Intimal and medial areas were compared and cell density was determined with fluorescent nuclear (Bisbenzimide) staining. Differential light microscopy with pentachrome staining was performed to determine the relative percent composition of intimal matrix constituents by stereological morphometric (point-count) techniques. RESULTS: The intimal areas for control and stenotic vein segments were 1.64 x 10(6) microm2 and 3.85 x 10(6) microm2, P < 0.0001, whereas the intimal nuclear densities (cells/unit volume) were 1.42 x 10(3) and 1.70 x 10(3) cells/microm2, P = 0.03. respectively. The corresponding medial area and medial nuclear densities were 5.01 x 10(6) microm2, 3.31 x 10(6) microm2; P = 0.08, and 2.27 x 10(3), 3.29 x 10(3); P = 0.001, for control and stenotic specimens, respectively. The intima:media area ratios were much greater, whereas the intimal and medial cell densities were only slightly greater in the stenotic compared with control veins. The relative composition of intimal extracellular matrix proteins of stenotic vein graft segments consisted of 21% cellular (fibrous) material, 33% collagen, and 46% glycosaminoglycan ground substance. CONCLUSION: The intimal lesions responsible for lower extremity vein graft stenosis are more hypertrophic than hyperplastic. Therapies aimed at preventing arterial and vein graft restenosis may thus need to inhibit matrix biosynthetic processes in addition to cellular proliferation.

Aged↗

Extracellular matrix distribution and islet morphology in the early postnatal pancreas: anomalies in the non-obese diabetic mouse.

Previously, we reported elevated numbers of macrophages in the pancreas of NOD mice, a spontaneous animal model for T1D, during the early postnatal period. Extracellular matrix plays an important role in the tissue trafficking and retention of macrophages as well as in postnatal pancreas development. Therefore, we have examined the expression and distribution of laminin and fibronectin, two major extracellular matrix proteins and their corresponding integrin receptors, in the pre-weaning pancreases of NOD mice and control mouse strains. In addition, we have characterized the pancreas morphology during this period, since the morphology of the pre-weaning pancreas before the onset of lymphocytic peri-insulitis, when the pancreas is still subject to developmental changes, has been poorly documented. We show that laminin labeling is mainly associated with exocrine tissue, whereas fibronectin labeling was mostly localized at the islet-ductal pole, islet periphery and in intralobular septa. Moreover, the protein expression level of fibronectin was increased in NOD pancreases at the early stage of postnatal development, as compared to pancreases of C57BL/6 and BALB/c mouse strains. Interestingly, pancreatic macrophages were essentially found at sites of intense fibronectin labeling. The increased fibronectin content in NOD neonatal pancreas coincided with altered islet morphology, histologically reflected by enlarged and irregular shaped islets and increased percentages of total endocrine area as compared to that of control strains. In conclusion, increased levels of the extracellular matrix protein fibronectin were found in the early postnatal NOD pancreas, and this is associated with an enhanced accumulation of macrophages and altered islet morphology.

Animals↗

Altered integration of matrilin-3 into cartilage extracellular matrix in the absence of collagen IX.

The matrilins are a family of four noncollagenous oligomeric extracellular matrix proteins with a modular structure. Matrilins can act as adapters which bridge different macromolecular networks. We therefore investigated the effect of collagen IX deficiency on matrilin-3 integration into cartilage tissues. Mice harboring a deleted Col9a1 gene lack synthesis of a functional protein and produce cartilage fibrils completely devoid of collagen IX. Newborn collagen IX knockout mice exhibited significantly decreased matrilin-3 and cartilage oligomeric matrix protein (COMP) signals, particularly in the cartilage primordium of vertebral bodies and ribs. In the absence of collagen IX, a substantial amount of matrilin-3 is released into the medium of cultured chondrocytes instead of being integrated into the cell layer as in wild-type and COMP-deficient cells. Gene expression of matrilin-3 is not affected in the absence of collagen IX, but protein extraction from cartilage is greatly facilitated. Matrilin-3 interacts with collagen IX-containing cartilage fibrils, while fibrils from collagen IX knockout mice lack matrilin-3, and COMP-deficient fibrils exhibit an intermediate integration. In summary, the integration of matrilin-3 into cartilage fibrils occurs both by a direct interaction with collagen IX and indirectly with COMP serving as an adapter. Matrilin-3 can be considered as an interface component, capable of interconnecting macromolecular networks and mediating interactions between cartilage fibrils and the extrafibrillar matrix.

Animals↗

Vascular smooth muscle cell phenotype influences glycosaminoglycan composition and growth effects of extracellular matrix.

Rat neonatal and neointimal vascular smooth muscle cells differ dramatically from adult medical vascular smooth muscle cells in their growth properties, with the neonatal and neointimal cells exhibiting growth in the absence of exogenously added growth factors. Since it has been hypothesized that extra-cellular matrix proteoglycans may influence the growth and differentiation of vascular smooth muscle cells, we examined the ability of matrix derived from these cells to influence vascular smooth muscle cell proliferation. To produce test matrices, cells were grown to confluence and removed by brief alkali treatment. Test cells were seeded onto these matrices and the rates of growth in a growth-factor-deficient medium determined. Compared to plastic wells, matrix from neonatal or neointimal cells stimulated the growth of vascular smooth muscle cells. Interestingly, matrix from adult cells was less efficient at promoting growth. Enzymatic digestion of extracellular matrix heparan sulfate, but not of other glycosaminoglycans, further increased the growth-stimulatory effect of extracellular matrix, suggesting that matrix heparan sulfate acts as a growth inhibitor. Consistent with this, biochemical analysis showed that the adult matrix contained a higher percentage of heparan sulfate compared with neonatal or neointimal matrix. These results suggest that autocrine production of heparan sulfate proteoglycans may play an important role in growth regulation of vascular smooth muscle cells during normal vascular development and differentiation as well as in pathological response to injury.

Age Factors↗

Specific inhibition of T-cell adhesion to extracellular matrix and proinflammatory cytokine secretion by human recombinant galectin-1.

The migration of immune cells through the extracellular matrix (ECM) towards inflammatory sites is co-ordinated by receptors recognizing ECM glycoproteins, chemokines and proinflammatory cytokines. In this context, galectins are secreted to the extracellular milieu, where they recognize poly-N-acetyllactosamine chains on major ECM glycoproteins, such as fibronectin and laminin. We investigated the possibility that galectin-1 could modulate the adhesion of human T cells to ECM and ECM components. T cells were purified from human blood, activated with interleukin-2 (IL-2), labelled, and incubated further with intact immobilized ECM and ECM glycoproteins in the presence of increasing concentrations of human recombinant galectin-1, or its more stable, related, C2-S molecule obtained by site-directed mutagenesis. The presence of galectin-1 was shown to inhibit T-cell adhesion to intact ECM, laminin and fibronectin, and to a lesser extent to collagen type IV, in a dose-dependent manner. This effect was specifically blocked by anti-galectin-1 antibody and was dependent on the lectin's carbohydrate-binding properties. The inhibition of T-cell adhesion by galectin-1 correlates with the ability of this molecule to block the re-organization of the activated cell's actin cytoskeleton. Furthermore, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production was markedly reduced when IL-2-activated T cells were incubated with galectin-1 or its mutant. This effect was prevented by beta-galactoside-related sugars. The present study reveals an alternative inhibitory mechanism for explaining the suppressive properties of the galectin-1 subfamily on inflammatory and autoimmune processes.

Cell Adhesion↗

The extracellular matrix as a scaffold for tissue reconstruction.

The extracellular matrix (ECM) consists of a complex mixture of structural and functional proteins and serves an important role in tissue and organ morphogenesis, maintenance of cell and tissue structure and function, and in the host response to injury. Xenogeneic and allogeneic ECM has been used as a bioscaffold for the reconstruction of many different tissue types in both pre-clinical and human clinical studies. Common features of ECM-associated tissue remodeling include extensive angiogenesis, recruitment of circulating progenitor cells, rapid scaffold degradation and constructive remodeling of damaged or missing tissues. The ECM-induced remodeling response is a distinctly different phenomenon from that of scar tissue formation.

Animals↗

Signaling through CD50 (ICAM-3) stimulates T lymphocyte binding to human umbilical vein endothelial cells and extracellular matrix proteins via an increase in beta 1 and beta 2 integrin function.

Regulated adhesion of T lymphocytes to antigen-presenting cells, endothelial cells and extracellular matrix proteins is crucial in T lymphocyte activation and migration to the sites of injury. In this study, we show that three monoclonal antibodies (mAb) recognizing different epitopes on the CD50 (ICAM-3) molecule increase T lymphocyte adhesion to tumor necrosis factor (TNF)-stimulated human umbilical vein endothelial cells and extracellular matrix proteins. These phenomena are mediated by an increase in beta 1 and beta 2 integrin avidity since (a) CD50-induced adhesion to endothelial cells was abrogated by simultaneous blocking of beta 1- and beta 2-mediated adhesion pathways but not by interfering with either one individually, (b) CD50 mAb increased beta 1 integrin-mediated adhesion to extracellular matrix proteins and to fibronectin-derived synthetic peptides, (c) CD50 mAb enhanced T lymphocyte binding to ICAM-1 transfectants, and (d) CD50 mAb did not modify surface expression patterns of beta 1 or beta 2 integrins on T lymphocytes. Our data suggest that constitutively expressed CD50 (ICAM-3) can play a pivotal role in initiating a cascade of adhesion events which may be crucial in immune activation and in the development of inflammatory lesions.

Antigens, CD↗

Inducible nitric oxide synthase in human lymphomononuclear cells activated by synthetic peptides derived from extracellular matrix proteins.

Synthetic peptides with sequences present in extracellular matrix proteins are capable of causing the expression of the inducible form of nitric oxide synthase (iNOS), detected by immunocytochemistry, and the release of NO by human lymphomononuclear cells incubated in their presence. Active peptides are 15-mers containing a characteristic 2-6-11 motif in which the amino acid residue at position 2 is Leu, Ile, Val, Gly, Ala or Lys; the residue at position 6 is always Pro; and residue 11 is Glu or Asp. The induction of iNOS in human monocytes and macrophages could be involved in the cytotoxicity against tumor cell lines also elicited by these peptides.

Amino Acid Oxidoreductases↗

Binding of hormonal steroids to isolated oligodendroglia and astroglia grown in vitro on a naturally produced extracellular matrix.

The ability of glial cells, grown on extracellular matrix (ECM), to bind corticosterone (CS), dexamethasone (Dex), and testosterone (T) was investigated. Isolated oligodendrocytes were able to specifically bind CS as well as T. Bound [3H]CS was partially displaced by excess T, whereas no displacement of [3H]T was observed with excess CS. Oligodendrocytes were not able to bind [3H]Dex. Astrocytes derived from hippocampal or hypothalamic slices showed no binding of either [3H]CS or [3H]Dex. The results demonstrate that ECM-grown oligodendrocytes possess receptors for CS and T but not for Dex. Furthermore, the binding sites for CS were less specific than the comparable sites for T. These results substantiate the notion that oligodendrocytes are involved in the action of steroid hormones within the brain.

Animals↗

Gelatin fragments block adherence of Candida albicans to extracellular matrix proteins.

The adherence of Candida albicans to extracellular matrix proteins may be a critical step in the pathogenesis of candidiasis. Yeast cell adherence to type I and IV collagen, fibronectin and laminin was blocked by peptide fragments from denatured type I collagen (gelatin). Gelatin fragments were obtained by digestion of the reduced protein with trypsin or CNBr. The fragments did not have antifungal properties, presumably inhibiting adherence by blocking receptors (adhesins) on the surface of the fungus. A 10-mer (GQRGVVGLPG) fashioned from the alpha-1 chain of type I collagen reduced adherence by 68%. However, a gelatin peptide possessing 47 amino acids reduced fungal adherence to type I collagen by 100%. Peptides derived from the biocompatible protein gelatin, therefore, may have a potential role in reducing the adherence of the fungus to host proteins.

Amino Acid Sequence↗

Bone extracellular matrix stimulates invasiveness of estrogen-responsive human mammary MCF-7 cells.

Bone is the most frequent site of metastasis in breast cancer. This causes destructive osteolytic lesions. To achieve metastasis to bone, breast cancer cells must proliferate in a new microenvironment, arrest on extracellular matrix and invade. Breast cancer cells progress in the invasive processes only if they destroy bone with the assistance of osteoclasts. In this work, we present data suggesting that MCF-7 cells, an estradiol receptor-positive cell line that exhibits modest invasive capacity, proliferate in the presence of soluble factors secreted by the osteogenic cell line SaOS-2. The cells acquire a more aggressive phenotype when cultured on an extracellular matrix produced by the same osseous cell line. Acquisition of the invasive phenotype appears to be related to the capacity of bone extracellular matrix to induce the expression of urokinase-like plasminogen activator by MCF-7 cells, which is specific for MCF-7 cells, given that MDA-231 cells, an estradiol receptor-negative and more aggressive cell line, did not show significant changes when cultured in the presence of soluble and insoluble bone factors.

Bone Neoplasms↗