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PubMed2002

Differentiation-dependent induction of CYP1A1 in cultured rat small intestinal epithelial cells, colonocytes, and human colon carcinoma cells: basement membrane-mediated apoptosis.

Rat small intestinal epithelial cells and human colon adenocarcinoma cells cultured on Matrigel expressed the differentiation specific enzyme, sucrase-isomaltase, as determined by indirect immunofluorescence. Rat small intestinal epithelial cells, rat colonocytes, and human colon adenocarcinoma cells developed an altered morphology when cultured on Matrigel and became apoptotic within 24-48 h. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin caused a 2- and 5-fold induction, respectively, of ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on plastic was not detected. 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment caused a 14-fold induction of transfected, rat CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment induced ethoxyresorufin-o-deethylase activity by 6- and 1.6-fold, respectively in rat colonocytes cultured on Matrigel. Induction of ethoxyresorufin-o-deethylase activity was not observed in rat colonocytes cultured on plastic. CYP1A1-promoter-luciferase activity was induced 3-fold by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat colonocytes cultured on Matrigel. Induction of CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells or rat colonocytes cultured on plastic was not observed. Ethoxyresorufin-o-deethylase activity in human colon adenocarcinoma cells, cultured on either plastic or Matrigel, was induced 7-fold by benzo[a]pyrene. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity was 2-fold greater in human colon adenocarcinoma cells cultured on Matrigel compared to cells cultured on plastic. Extracellular matrix-mediated differentiation and apoptosis of intestinal cells provide in vitro systems for study of the regulation of CYP1A1 expression, carcinogen activation in the gut and mechanism(s) of apoptosis of colon cancer cells.

Animals↗
PubMed1987

Reconstituted basement-membrane matrix modulates fibroblast activities in vitro.

Cellular growth and collagen biosynthesis were compared in dermal calf fibroblasts cultured on plastic or on a reconstituted basement membrane gel, termed matrigel. This matrix, extracted from Engelbreth-Holm-Swarm tumors, consists mainly of laminin, entactin, type IV collagen, and heparan sulfate proteoglycan. The multiplication rate of fibroblasts grown on matrigel was stimulated compared to that of monolayered cells cultured on plastic, and these cells formed multilayers after 4 days. Protein and collagen biosynthesis was reduced in fibroblasts cultured on matrigel. A higher proportion of the newly synthesized collagen (40%) was incorporated to the extracellular matrix in cultures grown on matrigel than in those grown on plastic (14%). Type III collagen was the preferential collagen type deposited on matrigel, and the ratio of type III:type I collagens secreted in the medium was also slightly higher in cultures grown on matrigel. Partially processed collagen was more abundant in fibroblasts grown on matrigel than in cells cultured on plastic. Finally, cells grown on matrigel exhibited a higher catabolic activity than cells grown on plastic. In this experimental model, the reconstituted basement-membrane matrix seems to influence the activities of fibroblasts significantly.

Animals↗
PubMed2000

Inhibition of invasion and experimental metastasis of murine melanoma cells by human soluble thrombomodulin.

Thrombomodulin (TM) is an anticoagulant molecule expressed on the endothelial cell surface and soluble TM antigen, which is present in human plasma and urine, represents the products of limited proteolytic cleavage of cell-surface TM. Recently, it was demonstrated that TM is also expressed on the surface of several tumor cells and the expression level of TM negatively correlated with malignancy in cancer. We investigated the effect of soluble TM isolated from human urine (uTM) on the invasion and metastasis of murine melanoma cells (B16F10 cells) through a reconstituted basement membrane (Matrigel) and in a murine model of experimental lung metastasis. Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in a dose-dependent manner in a range from 10 to 1000 ng/ml uTM as compared with the control Matrigel without uTM. The inhibitory action of uTM was not altered in the presence of an excess amount of hirudin, an inhibitor of thrombin proteolytic activity, but abolished in the presence of anti-human TM IgG. Matrigel reconstituted with thrombin (1 NIH unit/ml) enhanced the invasion level of cells by 1.5-fold relative to the control Matrigel without thrombin. The thrombin-enhanced invasion of B16F10 cells was repressed by addition of hirudin (10 units/ml) or uTM (100 ng/ml) into the Matrigel. Matrigel reconstituted with hirudin (10 units/ml) and uTM (100 ng/ml) additionally accelerated the inhibitory activity of hirudin or uTM on the thrombin-enhanced invasion of B16F10 cells. Moreover, metastatic colonies formed in the lungs of mice injected intravenously with B16F10 cells were significantly reduced by injection of uTM once a day up to 2 days after co-injection of uTM with the cells. These results suggested that Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in vitro through a thrombin-independent mechanism and the injection of uTM suppressed experimental lung metastasis of the cells in mice.

Animals↗
PubMed2001

Comparison of three in vitro human 'angiogenesis' assays with capillaries formed in vivo.

Angiogenesis assays are an important tool for studying both the mechanisms of angiogenesis and the potential development of therapeutic strategies to modulate neovascularisation. In vivo angiogenesis assays are considered to be the most informative of these but are often expensive, time-consuming and require specialist training to perform. In vitro assays tend to be more rapid, less expensive and easier to interpret. In vitro angiogenesis assays operate on the principle that endothelial cells form tubule-like structures when cultured on a supportive matrix. Assays involving a matrix derived from murine tumours, Matrigel (or a growth factor reduced form of this), are now the most common in vitro tubule formation assays. However, another tubule formation assay has recently been developed in which endothelial cells are co-cultured with fibroblasts. Here, we have used quantitative image analysis to compare the morphological features of tubules formed in the Matrigel assay and this new 'Co-culture' assay, with those of capillaries formed in a microvascular bed in vivo. Tubules formed in standard and growth factor reduced Matrigel assays were short and relatively homogeneous, whereas those formed in the Co-culture assay were significantly more heterogeneous, consisting of both short and long interconnecting tubules that more closely resembled capillaries than Matrigel tubules. Moreover, cells on Matrigel, and to a lesser extent growth factor reduced (GFR) Matrigel, often clumped into large cell aggregates, a feature rarely seen in the Co-culture assay. In addition, we demonstrate that Matrigel stimulates tubule formation by various non-endothelial cell types, suggesting that tubule formation by endothelial cells may not represent true differentiation of this cell type. In summary, the morphology of tubules in the Co-culture assay appears more representative of capillary formation in vivo, than the endothelial cell changes that occur in either form of Matrigel assay.

Animals↗
PubMed2002

Migration through basement membrane modulates eosinophil expression of CD44.

BACKGROUND: Tissue eosinophils express more membrane receptors and release more mediators than blood eosinophils, suggesting that migration from blood to tissue modulates eosinophil phenotype and functions. OBJECTIVE: We postulated that eosinophil passage through endothelial basement membrane, an important step of eosinophil migration into tissue, may be responsible for some of these changes. METHOD: We previously showed that 5-oxo-6, 8, 11, 14-eicosatetraenoic acid (5-oxo-ETE) in combination with IL-5 promotes eosinophil migration through Matrigel, a mouse tumour cell-derived basement membrane. Using this model, we evaluated the effect of trans-Matrigel migration on purified human blood eosinophil expressions of CD44, CD69 and HLA-DR that either increase or appear on activated eosinophils, and releases of peroxidase (EPO), leukotriene (LT) C(4) and granulocyte-monocyte colony stimulating factor (GM-CSF). RESULTS: IL-5, but not 5-oxo-ETE, increased eosinophil expression of CD44 and CD69. Migration of eosinophils through Matrigel significantly increased CD44 expression level over the one induced by IL-5 (P = 0.0001). Migration through Matrigel did not modify CD69 expression compared with the one obtained in the presence of IL-5 alone; however, incubation of eosinophils on Matrigel decreased IL-5-induced CD69 (P = 0.0001). Trans-Matrigel migration did not modify HLA-DR expression, nor EPO, LTC(4) and GM-CSF releases. CONCLUSION: These data show that in vitro trans-Matrigel migration and Matrigel contact modulate eosinophil membrane receptor expression. Consequently, they suggest that migration through basement membrane mediates changes in cell-surface phenotype observed on activated eosinophils and probably prepares them for interactions with tissue components and cells.

Adolescent↗
PubMed1996

Laminin mediates basement membrane induced differentiation of HEC 1B endometrial adenocarcinoma cells.

In vitro studies on endometrial carcinogenesis have been hampered by limited differentiation of the cells in culture. Using the endometrial carcinoma cell lines HEC 1B and its subclone HEC 1B(L), we established and characterized cell culture conditions that preserve a more differentiated state of the tumor cells. Randomly seeded HEC 1B(L) cells, if grown in a serum-free defined medium on top of a reconstituted basement membrane (Matrigel), within a few hours assembled themselves to web-like structures. In a thick layer of Matrigel, they showed an even more pronounced morphological differentiation. Functionally, two additional secretory proteins, about 31 and 77 kDa in size, became apparent as a response to matrigel. To further investigate the regulatory role of the extracellular matrix in the process of in vitro differentiation of endometrial adenocarcinoma cells, we addressed two specific problems. First, we investigated if the capacity of in vitro differentiation is a specific feature of HEC 1B(L) cells or if it is common to all endometrial adenocarcinoma cells. Second, we tried to identify the Matrigel component(s) responsible for in vitro differentiation. The assembly of HEC 1B and HEC 1B(L) cells into spatially organized web-like structures and the expression of the 77 kDa protein were thereby used as an assay. All endometrial adenocarcinoma cell lines tested to a variable degree formed web-like structures on Matrigel. Although the pattern of de novo synthesized secretory proteins changed as a response to Matrigel, only HEC 1A, HEC 1B, HEC 1B(L), and Ishikawa cells responded to culture on Matrigel by an increased expression of the 77 kDa protein. Functionally, polyclonal anti-laminin antibodies, but not anti-collagen type IV antibodies, disrupted formation of web-like structures by HEC 1B cells. The laminin-specific peptides YIGSR and SIKVAV but none of the RGD-peptides RGDS, GRGDSP, or GRADSP affected the three-dimensional assembly of these cells in vitro. Both anti-laminin antibodies and laminin-specific peptides suppressed Matrigel-induced formation of the 77-kDa secretory protein by HEC 1B cells. These findings suggest the involvement of laminin in the in vitro differentiation of the HEC 1B endometrial adenocarcinoma cell line. In a mechanistic view, laminin appears to play a crucial role in the regulation of this in vitro differentiation process.

Adenocarcinoma↗
PubMed1992

Expression of 11 beta-hydroxylase and 21-hydroxylase in long-term cultures of bovine adrenocortical cells requires extracellular matrix factors.

In order to elucidate mechanisms for the loss of expression of 11 beta-hydroxylase and 21-hydroxylase, induction of these genes in long-term cultures of bovine adrenocortical cells was reassessed and compared with induction of 17 alpha-hydroxylase. We previously showed that both 11 beta- and 21-hydroxylases require insulin-like growth factor-I (IGF-I) as well as cAMP for induction; these are the only factors needed by primary cultures. Cells at population doubling level 10 grown on fibronectin-coated polystyrene dishes and incubated with cholera toxin and IGF-I did not express 11 beta-hydroxylase and 21-hydroxylase. They showed a truncated steroidogenic pathway, converting 25-hydroxycholesterol to some 11-deoxycortisol but little cortisol. However, when population doubling level 10 cells were grown for 5 days in extracellular matrix Matrigel, cholera toxin and IGF-I induced a complete steroidogenic pathway to cortisol. Northern blotting also showed that expression of 11 beta-hydroxylase messenger RNA (mRNA) after cholera toxin/IGF-I induction was observed only in cultures grown in Matrigel and was undetectable in cultures grown on plastic. 21-Hydroxylase mRNA was observed in cultures grown on plastic but was greatly enhanced by Matrigel; however, 17 alpha-hydroxylase mRNA was induced to a similar extent with and without Matrigel. In other middle passage cultures, whether grown as mass cultures, SV40 T antigen-transfected clones, or normal (nontransfected) clones, cells did not express 11 beta-hydroxylase except when grown in Matrigel; 21-hydroxylase was low and expression was enhanced by Matrigel, whereas 17 alpha-hydroxylase expression was unaffected. As previously determined, in late-passage cells and clones only side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase activities were detected and 17 alpha-hydroxylase was not expressed. In such cells 11 beta-hydroxylase and 21-hydroxylase were also not expressed, even in the presence of Matrigel. Thus, prior to the previously described loss of expression of 17 alpha-hydroxylase, Matrigel permits the cholera toxin/IGF-I-induced expression of a complete steroidogenic pathway in bovine adrenocortical cells in long-term culture.

Adrenal Cortex↗
PubMed2001

Effect of the extracellular matrix on pancreatic endocrine cell function and its biocompatibility in dogs.

The effect of the synthetic extracellular matrix (ECM) in a diffusion chamber for a bioartificial endocrine pancreas (Bio-AEP) on pancreatic endocrine cells in vitro and its biocompatibility in dogs were investigated. Two different types of ECM were used: type I collagen treated with low antigen (type I LA), and reconstituted basement membrane matrix (Matrigel) derived from Englbreth-Holm-Swarm (EHS) mouse sarcoma. Matrigel contains growth and differentiation factors and cell adhesion molecules such as laminin, heparan sulfate proteoglycan, and entactin. Purified porcine pancreatic endocrine (PE) cells were suspended in type I LA or Matrigel and then placed into a 12-well culture plate (4 x 10(7) cells/ml gel/well). The insulin accumulation from PE cells in Matrigel was significantly greater than that in type I LA (9.3 +/- 3.6 mU/well vs. 2.3 +/- 1.3 mU/well). When Bio-AEP with Matrigel and PE cells was implanted into the abdominal cavity of a pancreatectomized diabetic dog, the exogenous insulin requirement for maintaining normoglycemia was reduced for the first 4 weeks. However, after 6 weeks of implantation, fasting blood glucose levels suddenly increased. Laparotomy revealed encapsulated Bio-AEP with thick fibrous tissue. Following removal of the Bio-AEP from the abdominal cavity, another Bio-AEP containing type I LA and PE cells was implanted into the same dog. The exogenous insulin requirement was gradually decreased to almost half that of preimplantation levels. Bio-AEPs containing type I LA or Matrigel, but not PE cells, were implanted into the abdominal cavities of four healthy dogs. After 4 weeks of implantation, the Bio-AEP with Matrigel was encapsulated with fibrous tissue similar to that in the diabetic dog, but the Bio-AEP with type I LA was not. These results indicate that Matrigel may be incompatible with dogs and that the type I LA is more suitable for Bio-AEP.

Animals↗
PubMed1992

Basement membrane and its components on lymphocyte adhesion, migration, and proliferation.

During inflammation and recirculation, lymphocytes migrate into tissues by traversing the capillary endothelium, a process known as extravasation. After crossing the endothelial cells, lymphocytes come into contact with the basement membrane, which is a specialized layer of extracellular matrix containing predominantly laminin, collagen type IV, entactin, and heparan sulfate proteoglycans. In tissue invasion by inflammatory cells and metastatic tumor cells, the basement membrane serves as a substratum for cell adhesion and migration. However, the role of basement membrane in lymphocyte extravasation remains unclear. In this study, we investigated the effect of basement membrane on lymphocyte adhesion, migration, and proliferation, using matrigel as a model for basement membrane. We observed that matrigel promotes both lymphocyte adhesion and migration, with entactin primarily responsible for promoting adhesion and laminin for promoting migration. In addition, activation of lymphocytes by anti-CD3 enhances their adhesion and migration on matrigel-coated substratum. We also observed that matrigel inhibits the proliferation of lymphocytes stimulated by Con A. Furthermore, we demonstrated that laminin is the matrigel component responsible for inhibiting lymphocyte proliferation. However, matrigel has no effect on the proliferation of lymphocytes stimulated by LPS. These results suggest that matrigel has different effects on lymphocyte subpopulations. In agreement with the results on proliferation, matrigel also inhibits the production of IL-2 by Con A-stimulated lymphocytes.

Animals↗
PubMedDate not supplied

Invasiveness and chemotactic activity of oncogene transformed NIH/3T3 cells.

The role of oncogenes in the acquisition of invasive and metastatic capabilities is controversial. Interactions with basement membranes are critical in the process of tumor invasion and metastasis. We compared the ability of 3T3 cells transformed by oncogenes involved in various stages of signal transduction to invade a reconstituted basement membrane in vitro and to grow in a three dimensional basement membrane gel (matrigel). Cell lines transformed by various oncogenes and oncoviruses: v-sis (a growth factor), v-erb-B (a truncated EGF receptor), Moloney sarcoma virus (v-mos: a protein kinase homologue), mutated c-ras oncogenes (G protein homologues), FBJ virus (v-fos: a nuclear protein) were investigated. All transformed cell lines were able to invade in the chemoinvasion assay, where a layer of matrigel is coated onto chemotaxis filters. FBJ/3T3 were the least invasive and SSV/3T3 the most invasive. Control 3T3 cells could not cross the matrigel barrier. All transformed cells grew on matrigel forming invasive, branching colonies, whereas control 3T3 were unable to grow in matrigel. Cells transfected with the v-erb-B gene grew as multilayers inside matrigel. Invasiveness and growth on matrigel were accompanied by a high chemotactic response to laminin (LN) in all transformed lines. These results suggest that invasion and growth on matrigel, together with migration to LN, are induced by a large spectrum of oncogenes. When 3T3 cells were transfected with v-sis oncogene under the transcriptional control of the metallothionein (MMT) promoter and exposed to Zn++, their in vitro invasiveness was specifically increased by around 3 fold. These findings provide further evidence supporting a direct role of the v-sis oncogene in the invasive phenotype.

Cell Transformation, Neoplastic↗
PubMed1994

A modified invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay for quantitating tumor cell invasion.

Reconstituted basement membrane matrix (Matrigel) has been utilized for in vitro assay of tumor cell invasion in recent years. In the conventional chamber for the invasion assay, however, a large number of cells passed easily through the center of the Matrigel-coated filter because the Matrigel layer could not be completely uniform by the meniscus formation. To prevent the meniscus phenomenon of the Matrigel layer, we devised a water-repellent treatment of the inside wall of the assay chamber with paraffin. Consequently, very few erythrocytes passed through the Matrigel-coated filter of this modified chamber with the erythrocyte assay, which was used to demonstrate the evenness and uniformity of the Matrigel layer on the filter. For quantitating a small number of cells which invaded through the Matrigel-coated filter by the invasion assay, a tetrazolium-based colorimetric 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay was used. The invasive abilities of the eight different cells were determined by this invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium+ ++ bromide assay using the modified chamber with a filter coated with 70 microliters of the 0.2-mg/ml Matrigel. After 72 h of incubation, the malignant cell lines significantly exceeded the normal cell lines in the percentage of invasion (P < 0.01). Therefore, the modified invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium+ ++ bromide assay provides a simple, easily reproducible in vitro assay for quantitating tumor cell invasion.

Animals↗
PubMed1998

Engraftment of chronic myeloid leukemia in SCID mice.

Chronic myeloid leukemia (CML) is a clonal disorder of primitive hematopoietic stem cells characterized by a reciprocal translocation between chromosomes 9 and 22. Animal models of CML would be useful to study the biology and potential therapies in this disease. Mice with severe combined immunodeficiency (SCID) which will accept human xenografts have been useful in the study of a variety of human malignancies. CML has been difficult to establish in SCID mice possibly due to the lack of a functioning human stroma and relevant cytokines. To facilitate engraftment we injected cells in matrigel which is a soluble extract of basement membranes; is liquid below 22 degrees C and gels at 37 degrees C. CD34+ myeloid blast crisis cells (2 x 10(6)) were mixed in matrigel and injected subcutaneously into 10 SCID mice. All mice developed large tumours which spread to the mouse BM and spleen. However the percentage of human cells in the mouse BM and spleen was variable and ranged from 1 to 50 per cent. In contrast chronic phase (CP) CML cells mixed in matrigel did not form subcutaneous tumours and spread to the BM and spleen was detectable by PCR and not macroscopically. Groups of mice were injected with matrigel containing 1-20 x 10(7) MNC (2-20 x 10(5) CD34+ cells) from five patients with CP CMP. Bcr-abl sequences were detected by RT-PCR in the peripheral blood (PB) of 38/84 (45 per cent) mice at 3-10 weeks following injection of the CML cells but rarely at later time points. In addition, 33/75 (44 per cent) of mice sacrificed between 7 and 35 weeks following injection of CML cells were bcr/abl positive in the bone marrow and 17/70 (24 per cent) were positive in the spleen. Bcr-abl positive human CFU-GM colonies were also cultured from the murine bone marrow of several mice indicating that hematopoietic progenitor cells were able to migrate from the matrigel and engraft in murine hematopoietic organs. Engraftment of CP-CML was more successful in mice given higher numbers of CD34+ cells. Histological examination revealed that myeloid cells grow locally in the matrigel for several weeks, during which time the matrigel is infiltrated by blood vessels which may allow for the migration of CML progenitors to the murine bone marrow. This model system may be useful for studying the role of immunotherapy after allogeneic and autologous bone marrow transplantation.

Adult↗
PubMed1997

Effects of extracellular matrix components on axonal outgrowth from peripheral nerves of adult animals in vitro.

Relatively little is known of the growth requirements for regenerating axons of the peripheral nervous system of adult animals. In the present study, we show that extracellular matrix material secreted by the Engelbreth-Holm-Swarm tumor cell line (matrigel) supports axonal growth from explanted peripheral nerve-dorsal root ganglia (DRG) preparations of adult mice and amphibia in serum-free media, without addition of growth factors. Axonal growth in matrigel was much more profuse than that in the more commonly used gels of type 1 collagen and, after some days in culture, was accompanied by migration of Schwann cells along axons. The most abundant protein in matrigel is laminin, which has been shown in many studies to support axonal growth but, surprisingly, antisera to laminin did not inhibit axonal growth in matrigel. To determine the ability of the major components of matrigel, laminin, type IV collagen, and heparan sulfate proteoglycan (HSPG), to support axonal growth, these proteins were added to preparations of mouse peripheral nerve-DRGs in type I collagen gels. Regenerating axons were significantly longer in the presence of laminin and type IV collagen than in control cultures, while HSPG had a slight inhibitory effect. In this assay system, however, diluted matrigel solution was even more effective in stimulating axonal growth than laminin or type IV collagen, either alone or in combination. The results suggest that in addition to laminin and type IV collagen, other components within matrigel may contribute to its ability to support axonal growth.

Ambystoma mexicanum↗
PubMed2003

Thioredoxin inhibits microvascular endothelial capillary tubule formation.

Thioredoxin (Trx) inhibited human HMEC-1 dermal microvascular endothelial cell capillary tubule forming capacity in a Matrigel based assay in vitro. Inhibition of capillary tubule formation was Trx catalytic site and thioredoxin reductase (TrxR) dependent, mediated at the Matrigel matrix level, and associated with a shift from morphological differentiation to continuous proliferation, with enhanced cell spreading resulting in eventual monolayer formation. Soluble complex carbohydrates, which inhibited capillary tubule formation on Matrigel without induction of cell spreading or monolayer formation, failed to impair Trx promotion of cell spreading and mono-layer formation, suggesting a shift away from carbohydrate-mediated cell/matrix adhesive interactions. Laminin peptides YIGRS and SIKVAV, which impaired tubule formation on Matrigel without inducing cell spreading or monolayer formation, partially impaired cell spreading upon Trx-treated Matrigel without restoring tubule formation, consistent with a potential role for laminin in Trx-mediated effects. Trx reduced laminin and destabilised laminin/galectin-3 complexes within Matrigel. Native purified EHS Laminin (also containing galectin-3), but not recombinant galectin-3, restored HMEC-1 capillary tubule formation on Trx-treated Matrigel. These data highlight a novel deregulatory effect of extracellular Trx upon morphological capillary differentiation that appears to depend upon the reduction of laminin and destabilisation of its interaction with galectin-3, possibly leading to galectin-3 neutralisation that shifts cell/matrix adhesive interactions away from being carbohydrate mediated and results in loss of proliferation-inhibiting and differentiation promoting cues from this tumor basement membrane matrix.

Capillaries↗
PubMed2000

Effect of extracellular matrix on the expression of peroxisome proliferation associated genes in cultured rat hepatocytes.

The purpose of this study was to determine whether the extracellular matrix used in hepatocyte culture would alter gene expression induced by the peroxisome proliferator ciprofibrate (CIP). We compared the activities and mRNA levels of two enzymes associated with peroxisome proliferation-fatty acyl-CoA oxidase (FAO), the first enzyme in the peroxisomal beta-oxidation pathway, and lauric acid hydroxylase (LAH), which represents the activity of the cytochrome P450 4A subfamily-in rat hepatocytes cultured on different plates: plastic, collagen-coated, thin and thick Matrigel, and collagen gel plates. CIP increased FAO activity about fivefold in collagen gel plates and sixfold in thick Matrigel plates compared to a fourfold increase in other plates; LAH was increased about threefold in thin Matrigel plates and fourfold in thick Matrigel and collagen gel plates compared to only a twofold increase in plastic and collagen-coated plates. The mRNA level for FAO was highest in hepatocytes cultured on collagen gel and Matrigel plates compared to those cultured on plastic and collagen-coated plates. The P-450 4A1 mRNA expression, however, was highest in the collagen gel plates, with lower expression in the thick Matrigel, collagen-coated and plastic plates. DNA synthesis and the DNA binding activity of the transcription factor AP-1 were also examined in response to epidermal growth factor (EGF) and CIP. Without the addition of EGF, DNA synthesis was significantly higher on collagen-coated plates than on collagen gel plates. The DNA binding activity of AP-1 was also induced after 24 hr culture in collagen-coated plates, whereas it was not detected in collagen gel plates. After the addition of EGF, the DNA binding activity of AP-1 was increased in both collagen-coated plates and collagen gel plates. CIP did not increase the DNA binding activity of AP-1 in either plate. These results demonstrate that components of the extracellular matrix influence the induction of peroxisome proliferator-induced enzyme activities and mRNA levels by CIP, with the highest induction seen in collagen gel and thick Matrigel plates. Furthermore, the induction of cell proliferation and AP-1 DNA binding activity are influenced by the extracellular matrix.

Acyl-CoA Oxidase↗
PubMed1993

Effect of reconstituted basement membrane components on the growth of a panel of human tumour cell lines in nude mice.

Previous reports have indicated that reconstituted basement membrane (matrigel), when co-injected with either established or primary human tumour cells, can improve the growth of subcutaneous xenografts in nude mice. The human adenocarcinoma cell lines A549, SW480, and WiDr, and the human fibrosarcoma cell line HT1080scc2 exhibit varying degrees of tumourigenicity in nude mice. All these lines showed increased tumorigenicity and/or growth rate, together with a change towards a more differentiated tissue morphology, when co-injected with matrigel into nude mice. Experiments using A549 cell line have indicated that the effect of matrigel is concentration-dependent and that increased growth rate is not maintained when xenografts grown with matrigel are passaged into further mice. These results strongly suggest that increased tumour growth results from the improved growth conditions afforded by matrigel, rather than from the selection of subpopulations of the most tumourigenic cells. Increased growth of intracaecal tumours arising from the co-injection of SW480 cells with matrigel, indicate a possible use for matrigel in the development of more relevant animal models using the orthotopic site. Purified laminin significantly increased the growth of sc tumours resultant from co-injection with either WiDr or A549 cells, whereas collagen IV or laminin with entactin showed no such effect. A role for free laminin in the stimulation of cell growth in the absence of an intact basement membrane is discussed.

Animals↗
PubMed1998

Differentiation of a colon cancer cell line on a reconstituted basement membrane in vitro.

Basement membrane, a thin extracellular matrix, functions as a tissue stabilizer that promotes tissue integrity and differentiated phenotype. We studied a human colon cancer cell line, SNU 61, to evaluate its ability to differentiate on basement membrane. Cells were cultured on plastic, reconstituted basement membrane (Matrigel) or polyhydroxyethyl methacrylate (poly HEMA) for 72 h and evaluated by light and electron microscopy. On Matrigel, the cells showed gland formation with highly polarized cells containing basal nuclei and well developed brush border microvilli on the luminal surface. Apoptosis was noted mainly at the luminal side. On electron microscopic examination, numerous long microvilli, abundant cytoplasmic organelles and intercellular junctions were noted in the Matrigel-cultured cells. Intermediate cytoskeletons were scattered in the cytoplasm and existed on the axes of microvilli. Junctional complexes and desmosomes were frequently formed along intercellular spaces. The cells cultured on poly HEMA, on the other hand, were poorly differentiated and contained a few glandular structures with small lumens. Brush border microvilli, characteristic of enterocytic differentiation, were few in number and were developed on the basal surface. Intermediate filaments and microtubules were fewer than in the Matrigel-cultured cells. Carcinoembryonic antigen was expressed on the luminal surface of the Matrigel-cultured cells and in the cytoplasm of the poly HEMA cultured cells. CD44 stained the basolateral surface in the Matrigel-cultured cells, but the basal side was not stained in the poly HEMA cultured cells. These results are consistent with the different localization of microvilli in the Matrigel and in the poly HEMA cultured cells. Our observations suggest that human colon cancer cells on basement membrane can undergo glandular differentiation and that extracellular matrix is an important factor in morphogenesis.

Basement Membrane↗
PubMed2002

Endogenous adipocyte precursor cells for regenerative soft-tissue engineering.

Subcutaneous injection of reconstituted basement membrane (Matrigel) in combination with basic fibroblast growth factor induces de novo adipogenesis in which endogenous precursor cells invade the artificially formed Matrigel space, proliferate and differentiate to form adipose tissue. Since this adipogenesis offers us a novel approach for soft-tissue reconstruction without transplanting preadipocytes, the early process was examined by optical and electron microscopy. Formation of multiple layers of fibroblast-like cells at the surface of Matrigel implant was the first response of connective tissue. The cells within four to five layers proximal to Matrigel implant acquired a thick cytoplasm and an enlarged nucleus, and they invaded Matrigel space together with endothelial cells which caused neovascularization. Phagocytotic incorporation and digestion of Matrigel components by well-developed lysosomes appeared to be a stimulus of fibroblast-like cells to mature depending on proximity to Matrigel. The fibroblast-like cells often contacted to the outer surface of capillary over a large area and rapidly accumulated lipid droplets. Electron microscopy of the developing adipocytes showed a well-organized smooth endoplasmic reticulum and mitochondria. This investigation thus revealed the characteristics of adipocyte precursor cells, which can be recruited for regenerative engineering of soft tissues.

Adipocytes↗