Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.     

Adhesion of human endometrium to the epithelial lining and extracellular matrix of amnion in vitro: an electron microscopic study

One of the first steps in the pathogenesis of endometriosis is the attachment of the endometrium to the peritoneal lining. Since the peritoneum is extremely fragile and hard to obtain, amnion has been used as an in-vitro model to study adhesion. Scanning and transmission electron microscopy was applied to evaluate the adhesion of endometrial cells isolated in the proliferative and secretory phases of the menstrual cycle. Endometrial fragments obtained in either phase of the cycle were able to adhere to the extracellular matrix of the amnion. Fragments from proliferative phase endometrium showed active spreading and growth over the matrix surface, whereas fragments from secretory phase endometrium did not. Fragments from proliferative as well as secretory phase endometrium were able to adhere to the epithelial side of the amnion, but only at locations where the amniotic epithelium was damaged or partly absent. These observations indicate that the basement membrane and extracellular matrix provide a suitable substrate for endometrial cell attachment and growth and that endometrial cell adhesion occurs preferentially to subepithelial structures, whereas an intact epithelium prevents the adhesion of endometrial fragments to the amnion.     

Altered deposition of extracellular matrix components in the skeletal muscle and lymph node of the MDX dystrophic mouse

1. MDX mice derived from a colony of C57BL/10ScSn mice develop an X-linked recessive muscular dystrophy, thus providing an adequate model to study the pathogenesis of muscular dystrophy. 2. Skeletal myofibers of MDX mutant mice were heterogeneous, with disorganization of myofilaments and the absence of immunolabelling for dystrophin with monoclonal antibody DY4/6D3. 3. Marked deposition of reticulin, collagenic fiber (types, I, IV) and laminin (LN) were consistently present mostly around lesioned and necrotic myofibers associated with an intense inflammatory reaction, whereas strong immunolabelling for TIII-C, TIV-C and FN was often associated with regenerated fibers. 4. During the onset (3 weeks of postnatal life) of disease and height of myonecrosis (5-6 weeks of postnatal life), popliteal lymph nodes showed dense argyrophilic meshwork, intense immunolabelling for collagens types I and IV, FN, LN and enlargement of the hili which were packed with mononuclear cells. Such alterations, albeit less intense, were still observed in MDX mice with 20 weeks of postnatal life. 5. The results support the view that ECM components might be influencing the migration of inflammatory cells and the process of myonecrosis in the skeletal muscle of MDX dystrophic mice.     

Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis

Interferon gamma (IFN-gamma) inhibits in vitro the activation of hepatic stellate cells (HSC), the primary extracellular matrix-producing cells in liver fibrosis. This study was undertaken to determine in vivo the effect of IFN-gamma in the rat model of liver fibrosis induced by dimethylnitrosamine (DMN), where HSC activation represents an early response to cell injury. Rats were killed after 1 or 3 weeks of treatment with DMN, IFN-gamma, DMN + IFN-gamma, or saline. Immunohistochemistry was used to identify proliferating (desmin-positive/bromodeoxyuridine (BrdU)-positive cells) and activated (alpha-smooth-muscle actin [alpha-SMA]-positive cells) HSCs. Collagen deposition was determined colorimetrically and by morphometry. The parenchymal extension of desmin- and actin-positive cells and of fibrotic tissue was measured by point-counting technique and expressed as a percentage of area. Western blot was used to determine laminin and fibronectin accumulation. The levels of messenger RNA (mRNA) for procollagen type I, fibronectin, and laminin were evaluated by Northern blot. No differences were observed in rats treated with either saline or IFN-gamma alone. IFN-gamma reduced HSC activation induced by liver injury, as shown by the decreased number of proliferating HSC and the reduction of parenchymal area occupied by alpha-SMA-positive cells observed in DMN + IFN-gamma-treated animals compared with the DMN group. This was associated with reduced collagen, laminin, and fibronectin accumulation and lower levels of mRNA for procollagen type I, fibronectin, and laminin in the DMN + IFN-gamma group. Thus, this study indicates that IFN-gamma reduces extracellular matrix deposition in vivo by inhibition of HSC activation.     

Complement activation occurs on subendothelial extracellular matrix in vitro and is initiated by retraction or removal of overlying endothelial cells

Vascular endothelium is continuously exposed to plasma complement, which could generate a potent proinflammatory signal if activated on the vascular wall. Normal endothelium, however, expresses an anti-inflammatory phenotype, which includes resistance to complement fixation. As activated endothelium converts to a proinflammatory phenotype, we investigated the effect of cytokines on endothelial susceptibility to complement fixation. Cytokine-treated HUVEC were exposed to human serum as a source of complement, and C3 deposition was quantified. IL-1beta and TNF-alpha in combination with IFN-gamma markedly increased endothelial C3 deposition; however, immunofluorescence microscopy revealed that the endothelial cells had retracted, and that bound C3 was concentrated not on cells but in areas of exposed subendothelial extracellular matrix (ECM). Studies with cell-free ECM indicated that complement activation required only ECM exposure and was independent of cellular activation. C3 deposition on ECM was reproduced by reconstituting the alternative pathway, which generated a stable C3 convertase on ECM, but not on endothelial cells. C3b and iC3b were identified on ECM exposed to purified alternative pathway components and serum, respectively. In conditions associated with endothelial disruption, exposure of subendothelial ECM could induce complement fixation and contribute to inflammation and vascular damage.     

Regulation of survival and death of mesangial cells by extracellular matrix

BACKGROUND: Cell-matrix interactions exert major effects on such phenotypic features as cell growth and differentiation. Apoptosis is an active form of cell death that is crucial for maintaining the appropriate number of cells as well as the organization of tissue. Recently, it has been suggested that apoptosis of the mesangial cells (MC) is important in glomerular remodeling after injury. The MC are surrounded by an extracellular matrix (ECM) in vivo. Since in disease conditions the mesangial matrix is altered quantitatively and qualitatively, it is of interest to determine whether cell-matrix interactions may influence apoptosis of the MC. METHODS: We first investigated the differences in the susceptibility to apoptotic stimuli of the MC cultured on various ECM components (type I collagen, fibronectin, basement membrane matrix). We then determined whether the inhibition of MC-matrix interactions would affect apoptosis. Finally, interactions between MC and matrix were disrupted by the inhibition of beta1-integrin expression with antisense oligonucleotides (ODN). RESULTS: When MC were cultured on type I collagen or fibronectin and deprived of serum for eight hours, the extracted DNA from the MC demonstrated an internucleosomal ladder pattern on gel electrophoresis that constituted the biochemical characteristic of apoptosis. However, no ladder pattern was apparent when MC were cultured on basement membrane matrix. The attachment of cells was completely inhibited when the MC were cultured on agarose-coated dishes for 24 hours. Gel electrophoresis of DNA extracted from these cells showed a ladder pattern. However, the MC attached to the substratum did not show any apoptosis. MC showed an increase in apoptotic cell death after treatment with antisense ODN against beta1-integrin molecule. CONCLUSIONS: These results indicate that normal ECM may prevent the MC from undergoing apoptosis and serve as a survival factor for MC. Signals from ECM that prevent apoptosis may be mediated by beta1-integrin molecules.     

Stimulation of extracellular matrix components in the normal brain by invading glioma cells

Malignant gliomas are characterized by an extensive invasion of tumor cells into the normal brain parenchyma. A substantial amount of data indicates that cell movement in general is regulated by specific interactions between extracellular matrix components and specific cell-surface receptors. In the present work, multicellular spheroids from 4 human glioma cell lines (U-373Mg, A-172Mg, U-251Mg and HF-66) were confronted with normal rat brain cell aggregates in vitro, which resulted in a progressive invasion of tumor cells into the brain aggregates. The co-cultures were then sectioned and immuno-stained for specific extracellular matrix components (laminin, fibronectin and collagen type IV) and for specific cell-surface receptors which bind to these components (integrins beta1, beta4, alpha3, alpha6). In addition, flow-cytometric measurements and Northern blot analyses showed expression of several different integrins within the cell lines. The alpha3 subunit was expressed strongly in all cell lines. Whereas the beta1 subunit was expressed weakly in exponentially growing monolayer cultures, it showed a pronounced expression in multicellular spheroids, indicating that the integrin expression may vary depending on the micro-environment within a tumor. Furthermore, normal brain tissue was able to produce laminin when confronted with the glioma cells, which also was observed for fibronectin and collagen type IV. The relevance of our observations to the in vivo situation was investigated further by immuno-staining 5 human glioma biopsy samples for laminin. In some areas of the tumors, specific deposits of laminin were observed. In conclusion, we have shown that normal brain tissue has the ability to produce extracellular matrix components, such as laminin, collagen type IV and fibronectin, when confronted with invading glioma cells. Our results show that the glioma cells express specific integrins which can interact with these extracellular matrix components. Such interactions may facilitate tumor cell migration and invasion.     

Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate

STUDY DESIGN: Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. OBJECTIVES: To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. SUMMARY OF BACKGROUND DATA: Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. METHODS: Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. RESULTS: Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. CONCLUSIONS: Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.     

Mechanochemical interactions between striated muscle cells of jellyfish and grafted extracellular matrix can induce and inhibit DNA replication and transdifferentiation in vitro

Striated muscle tissue of jellyfish was isolated with its adhering extracellular matrix (ECM) and cultured. Without further treatment the cultured muscle cells maintain their differentiated state. If, however, the isolated tissues are combined with cell-free ECM from the jellyfish or its polyp, DNA replication and proteolytic activity are induced followed by transdifferentiation into RF-amide-positive nerve cells. Changes in the mechanochemical interactions between the cells and the grafted ECM seem to induce the signals which lead to transdifferentiation. If the isolates are combined with small floating pieces of ECM most cells will leave their own ECM and overgrow the ECM graft. All cells in the combinations will then transdifferentiate. If the isolates are grafted onto large pieces of ECM kept permanently stretched on glass, a majority of cells will migrate onto the grafted ECM where they form a flat monolayer. In this case, however, DNA replication and transdifferentiation occurs mainly in those cells which have remained on or near their own ECM. Labeling experiments with [3H]-thymidine demonstrate that initiation of DNA replication occurs first in those cells which bridge from the native ECM to the grafted ECM. On the other hand inhibition of DNA replication and transdifferentiation is generally suppressed whenever tissues are allowed to form a monolayer of well-stretched cells. From these observations we conclude that mechanochemical interactions between the muscle cells and their substrate are responsible for both activation and inhibition of DNA replication and transdifferentiation.     

Function of proteoglycans in the extracellular matrix

Estimating phylogenies from DNA sequence data has become the major methodology of molecular phylogenetics. To date, molecular phylogenetics of the vertebrates has been very dependent on mtDNA, but studies involving mtDNA are limited because the several genes comprising the mt-genome are inherited as a single linkage group. The only apparent solution to this problem is to sequence additional genes, each representing a distinct linkage group, so that the resultant gene trees provide independent estimates of the species tree. There exists the need to find novel gene sequences which contain enough phylogenetic information to resolve relationships between closely related species. A possible source is the nuclear-encoded introns, because they evolve more rapidly than exons. We designed primers to amplify and sequence the 7 intron from the beta-fibrinogen gene for a recently evolved group, the woodpeckers. We sequenced the entire intron for 10 specimens representing five species. Nucleotide substitutions are randomly distributed along the length of the intron, suggesting selective neutrality. A preliminary analysis indicates that the phylogenetic signal in the intron is as strong as that in the mitochondrial encoded cytochrome b (cyt b) gene. The topology of the beta-fibrinogen tree is identical to that of the cyt b tree. This analysis demonstrates the ability of the 7 intron of beta-fibrinogen to provide well resolved, independent gene trees for recently evolved groups and establishes it as a source of sequences to be used in other phylogenetic studies.     

Relationship of an extracellular matrix protein, tenascin and breast diseases

Tenascin is an extracellular matrix glycoprotein consisting of six disulfide-linked subunits with molecular masses of 190-250 kDa. Molecular analysis of the tenascin gene revealed that it contains a region homologous to epidermal growth factor genes, repetitive sequences of the type III fibronectin and the fibrinogen gene. Culture studies have shown that tenascin has multiple functions including cell attachment and detachment, promotion and inhibition of neural crest cell migration, cell growth stimulation and hemagglutination. Immunohistochemically, tenascin shows a characteristic and spatially restricted distribution. In mouse mammary glands, tenascin protein is demonstrated in the dense mesenchyme present around growing epithelia during embryogenesis and oncogenesis. Tenascin is expressed in normal human adult breast tissue and benign conditions, although it is expressed more abundantly in breast cancer tissue. Prominent tenascin staining is found in dense cancer-mesenchymal junctions. The staining positivity is significantly correlated with metastasis to regional lymph nodes and tumor grade. Tenascin positive patients have a significantly poorer prognosis compared with tenascin-negative patients. Although the biological functions of tenascin in breast cancer tissue have not yet been clearly elucidated, tenascin staining in surgical tissue specimens might be useful when applied to detect a subgroup of breast cancer patients who have a poorer prognosis.     

Identification of biologically active sites in laminin an extracellular matrix protein

Laminin-1, a major component of basement membranes, has multiple biological activities including promotion of cell adhesion, spreading, migration, growth, neurite outgrowth and tumor metastasis. Several active sites on laminin-1 have been identified previously. We modified these biologically active peptides to enhance their activities. The multimeric YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides assembled on a branched lysine core were found to strongly enhance the activity of YIGSR in inhibiting tumor growth and metastasis. We also found the all-D-configuration peptide segment containing the IKVAV (Ile-Lys-Val-Ala-Val) sequence had similar biological activities to the native all-L-peptide in vitro and in vivo. These results suggest that these modified compounds are potentially useful for clinical applications. We have identified new active sequences from the laminin alpha 1 chain carboxyl-terminal globular domain (G domain). Using a systematic screening for cell binding sites with 113 overlapping synthetic peptides, we found five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. AG-10 and AG-32 were found to interact with integrins. AG-73 caused metastases of B16-F10 mouse melanoma cells to the liver colonization in mice. Additionally AG-73 was found to promote neurite outgrowth. Moreover, this peptide inhibited laminin mediated acinar-like development of a human submandibular gland cell line. The AG-73 domain on laminin-1 could be one of the most important biologically active sites. These active peptides may useful for study of the molecular mechanism of laminin-receptor interactions and for development of therapeutic reagents for tumor metastasis and angiogenasis.     

Function at the junction: dynamic interactions between lung cells and extracellular matrix

Recent advances are elucidating the mechanisms by which cells communicate with the surrounding matrix. Cells have specific receptors for matrix proteins. A number of intracellular molecules with signalling functions aggregate at specialised focal adhesion points and facilitate transfer of information both into and out of cells. The importance of these signaling processes to cell biology makes it likely that manipulation of these processes will allow innovative therapeutic approaches to lung disease.     

Nitric oxide modulates the synthesis of extracellular matrix proteins in cultured rat mesangial cells

Nitric oxide (NO) is an important effector molecule of the inflammatory response. It is synthesized by mesangial cells and has been proposed to contribute to glomerular injury in various disease states. We studied whether NO modulates extracellular matrix production in cultured rat mesangial cells. Stimulation of rat mesangial cell NO release with gamma-interferon and lipopolysaccharide resulted in reduced production of collagen (by 35%) fibronectin (by 48%) (P < 0.05). In contrast, laminin synthesis was enhanced two-fold by the same maneuver (P < 0.05). These changes were reversed by the addition of L-NAME, a selective inhibitor of inducible nitric oxide synthase. This is the first demonstration that NO regulates the synthesis of extracellular matrix by mesangial cells. The results indicate that increased renal production of NO in glomerular diseases may attenuate the production and accumulation of matrix proteins and limit the severity of glomerulosclerosis.     

Abnormal adhesion of T cells to extracellular matrix proteins in hemodialysis patients

MOTIVATION: The BioCatalog is a database of information on software of interest in molecular biology and genetics. The programs are grouped by domain of interest. AVAILABILITY: The BioCatalog is freely distributed as ASCII files. It can be searched through its Web interface at http://www.ebi.ac.uk/biocat. CONTACT: biocat@ebi. ac.uk     

Influence of extracellular matrix proteins on the development of cultured human dendritic cells

The development of dendritic cells (DC) is still only partly understood. Recently established culture systems using CD34+ cells or monocytes as precursor cells for the generation of DC indicate the necessity of pro-inflammatory cytokines for their development. In vivo the contact to other cells or to the proteins of the extracellular matrix might also be essential for their development. In our experiments we used granulocyte-macrophage colony-stimulating factor- and IL-4-treated human monocytes as precursor cells to investigate the interaction of DC at different maturation stages with the matrix proteins fibronectin, collagen type I and collagen type IV. We demonstrate a strong beta1-integrin-mediated adherence of immature DC to fibronectin that is lost completely during maturation. The binding to collagen type I was less strong but induced a maturation of the precursor cells. After 3 days of culture on this protein, the cells showed all features of fully matured DC such as expression of CD83 and an excellent allostimulatory capacity. The reason for this effect was shown to be the induction of TNF-alpha production by the DC themselves. In contrast to the adhesion to fibronectin, the maturation and the cytokine production of DC induced by collagen type I could not be inhibited by blocking of beta1-integrins. These results indicate that proteins of the extracellular matrix play an important role in the development and function of human DC.     

Regulation of alpha2-macroglobulin expression in rat Sertoli cells and hepatocytes by germ cells in vitro

The combination of an immunohistochemical technique and a panel of monoclonal antibodies was used to investigate the presence of leukocyte populations in the distal jejunal lymph node of 3-4 week old calves and adult cattle. The application of computer-assisted morphometric analysis enabled information to be obtained on the distribution of leukocyte populations in lymphoid compartments of the lymph node cortex. Semi-quantitative estimates of the areas of staining in histological sections showed that calves possessed significantly fewer B-cells and CD4+ cells in the outer cortex and significantly fewer T-cells (CD4+, CD8+ and gamma delta T-cells) in the deep cortex. These findings were interpreted to be a possible consequence of immunosuppression resulting from the passive transfer of maternal immunity in colostrum. The presence of some B-cell follicles in the region defined as the deep cortex suggested the on-going differentiation of this predominantly T-cell compartment. The larger presence of interdigitating cells (IDC) in the deep cortex of calves than adults was suggested by significantly larger CD1+ populations and it was argued that this could be the result of the confrontation with exogenous antigen faced by calves in early postnatal life. Antigen presenting populations, pan MHC II+ and MHC II DQ+ populations, were increased in all compartments of calf lymph nodes but were not significantly different from the populations in adult lymph nodes. Variance component analysis of the data generated in the present study showed that the image analysis technique was an effective and statistically powerful approach to investigate leukocyte populations within the specific microenvironments of the lymph node.     

Structure and biological activity of the extracellular matrix

The extracellular matrix is formed by complex and intricate networks within which molecules are precisely organized. These molecular networks determine the specific histoarchitecture of tissues and provide cells with information and a scaffold. Most of the structural extracellular matrix molecules - collagens, noncollagenous glycoproteins, and proteoglycans - are chimeric and share common domains. Studies of the interactions between extracellular matrix molecules and mapping of the interaction sites to defined structural modules have led to the concept that the function of the extracellular matrix relies largely in the polymers that they form. Furthermore, determination of the tertiary structure of protein motifs involved either in the assembly of the various molecules into polymers or in cell-extracellular matrix interactions has recently opened the field of structural biology of the extracellular matrix.