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PubMed2000

In vitro analysis of antiangiogenic activity of fungi isolated from clinical cases of equine keratomycosis.

OBJECTIVE: The goal of this project was to explore the possibility that fungal organisms produce metabolites that inhibit angiogenesis. Procedures Fungal cultures were obtained from cases of keratomycosis, grown in Sabouraud's dextrose broth, and sterile filtered for use in experiments. The Matrigel assay was used to screen the filtrate samples for antiangiogenic activity. Matrigel is a basement membrane matrix that supports the differentiation of human umbilical vein endothelial (HUVE) cells into a capillary-like network of tubules. HUVE cells were cultured using standard techniques and passaged at confluence, with all cells being used at passage 3-6. HUVE cells (40 000 cells) were pipetted into each well of a 24-well tissue-culture plate coated with Matrigel. An aliquot of fungal media filtrate was added to each well and the plates allowed to incubate for 18 h, at which time they were evaluated for tubule formation. RESULTS: Two fungal isolates showed inhibition of tubule formation. The addition of 100, 200 and 400 &mgr;L of the fungal media filtrate from the first isolate (Fusarium sp. 99A34574) produced a consistent and dose-dependent inhibition of tubule formation. The second isolate (Aspergillus sp. 271599) did not show inhibition of tubule formation with 100 or 200 &mgr;L added to the wells, however, it did show inhibition at 400 &mgr;L/well. The remaining three isolates did not cause inhibition at any concentration. CONCLUSIONS: Our findings suggest that certain fungal organisms produce metabolites that inhibit tubule formation in vitro, and that these metabolites may play a significant role in altering the host vascular response to fungal infections of the cornea.

Journal Article↗
PubMed2003

Microscopic analysis of the cellular events during scatter factor/hepatocyte growth factor-induced epithelial tubulogenesis.

Scatter factor/hepatocyte growth factor (SF/HGF), a large multifunctional polypeptide growth and motility factor, is known to play important roles during embryonic development, adult tissue growth and repair. In an established three-dimensional type I collagen model, SF/HGF induces Madin-Darby canine kidney (MDCK) epithelial cysts to form long, branching tubules (tubulogenesis). In addition, the composition of the surrounding extracellular matrix (ECM) has been shown to modulate SF/HGF-induced morphogenesis, where tubulogenesis was completely abrogated in Matrigel basement membrane. Many cellular events that occur during SF/HGF-mediated remodelling, and its modulation by the ECM, remain unclear. We have investigated these mechanisms through microscopic examination of the time-course of SF/HGF-induced responses in MDCK cysts cultured in type I collagen or Matrigel. We found that early responses to SF/HGF were matrix-independent. Changes included increased paracellular spacing between normally closely apposed lateral membranes, and the formation of filopodial processes, indicating a partial motile response. Cell-cell contact was maintained, with the persistence of cell junctions. Therefore, while one or a number of ECM components are preventing SF/HGF-primed cells from undergoing an invasive and/or migratory programme, non-permissive matrices are not preventing SF/HGF signalling to the cell. Later matrix-dependent responses, which occurred in type I collagen but not Matrigel, included the formation of basal protrusions that comprise two or more neighbouring cells, which extend to form nascent tubules. Modified polarity of cells comprising the basal protrusions was evident, with a marker for the apical membrane being found in the same region as adherens junctions and desmosomes, typically localized at lateral membranes. We propose a model for SF/HGF-induced tubulogenesis in which tubules form from basal protrusions of adjacent cells. This mechanism of in vitro tubule formation has many similarities to reported in vivo epithelial tubulogenesis.

Animals↗
PubMed2001

Autocrine motility factor enhances hepatoma cell invasion across the basement membrane through activation of beta1 integrins.

Autocrine motility factor/phosphohexose isomerase (AMF/PHI) is a cytokine that is linked to tumor invasion and metastasis. In hepatocellular carcinoma (HCC) tissues, hepatoma cells produce AMF/PHI and its receptor, Mr 78,000 glycoprotein (gp78), is strongly detected in hepatoma cells invading into the stroma and tumor thrombi in the portal vein. Here, we investigated the mechanism of hepatoma cell invasion through Matrigel induced by AMF/PHI using 3 hepatoma cell lines. Production of AMF/PHI, phosphorylation of MEK1/2, and Rho activity were investigated by immunoblotting. Expression of AMF/PHI and gp78 was observed by confocal fluorescence microscopy. The influence of AMF/PHI on activated integrin beta1 subunit expression was evaluated by flow cytometry. Changes in invasion, adhesion, and motility induced by AMF/PHI were evaluated using chemoinvasion, adhesion, and phagokinetic track motility assays. The effect of AMF/PHI on matrix metalloproteinase (MMP) secretion was evaluated by gelatin zymography. Hepatoma cells produced AMF/PHI and expressed gp78. Although AMF/PHI was ubiquitously detected, gp78 was strongly expressed in migrating cells. AMF/PHI induced up-regulation of activated integrin beta1 subunit expression. AMF/PHI stimulated hepatoma cell invasion through Matrigel, and stimulated the adhesion, motility, and MMP-2 secretion of hepatoma cells. The latter effects were suppressed by the function-blocking antibody for integrin beta1 subunit. AMF/PHI also enhanced Rho activity and the phosphorylation of MEK1 and MEK 2. Our results indicate that AMF/PHI enhances hepatoma cell invasion through Matrigel in an autocrine manner by stimulating the adhesion, motility, and MMP-2 secretion of these cells through activation of beta1 integrins.

Basement Membrane↗
PubMed2003

The alpha2 and alpha3 integrins are required for morphologic differentiation of an intestinal epithelial cell line.

BACKGROUND: The molecular mechanisms controlling intestinal epithelial cell differentiation are poorly defined because of the difficulty of growing normal intestinal cells. We have taken advantage of the ability of the Caco-2 cell line to acquire a glandular phenotype in 3-dimensional (3-D) culture systems to investigate the role of alpha2 and alpha3 integrins in morphologic differentiation. METHODS: Caco-2 cells transfected with sense or antisense DNA constructs of alpha2 or alpha3 integrins were grown in 3-D Matrigel or collagen I in the presence or absence of integrin function-blocking antibodies. We used light and confocal microscopy, BrDU incorporation, TUNEL assay, a fluorometric adhesion assay, FACS analysis, and Western blot analysis to study the effect of extracellular matrix (ECM) and integrins on morphology, polarization, proliferation, apoptosis, cell adhesion, and integrin expression. RESULTS: Compared to collagen I, Caco-2 cells cultured in 3-D Matrigel display cytoskeletal and adherens junction rearrangements and decreased proliferation consistent with cellular differentiation. These changes, which are inhibited by alpha2 and alpha3 blocking monoclonal antibodies and alpha2 and alpha3 antisense DNA transfection, were associated with an increase in alpha3 integrin expression. CONCLUSIONS: We demonstrated that signaling through both constitutively expressed alpha2 integrin and Matrigel-induced alpha3 integrin expression is required to acquire a differentiated phenotype in Caco-2 cells.

Antibodies, Monoclonal↗
PubMed2006

Platelets and platelet adhesion support angiogenesis while preventing excessive hemorrhage.

Platelets contain both pro- and antiangiogenic factors, but their regulatory role in angiogenesis is poorly understood. Although previous studies showed that platelets stimulate angiogenesis in vitro, the role of platelets in angiogenesis in vivo is largely uncharacterized. To address this topic, we used two in vivo approaches, the cornea micropocket assay and the Matrigel model, in four animal models: thrombocytopenic, Lyst(bg) (platelet storage pool deficiency), glycoprotein (GP) Ibalpha/IL4R transgenic (lacking extracellular GPIbalpha, the receptor for von Willebrand factor as well as other adhesive and procoagulant proteins), and FcgammaR(-/-) (lacking functional GPVI, the collagen receptor) mice. Adult mice were rendered thrombocytopenic by i.p. administration of an antiplatelet antibody. The number of growing vessels in the thrombocytopenic mice was lower in the cornea assay, and they showed significantly increased appearance of hemorrhage compared with mice treated with control IgG. The thrombocytopenic mice also showed more protein leakage and developed hematomas in the Matrigel model. GPIbalpha/IL4R transgenic mice presented increased hemorrhage in both assays, but it was less severe than in the platelet-depleted mice. FcgammaR(-/-) and Lyst(bg) mice showed no defect in experimental angiogenesis. Intravital microscopy revealed a >3-fold increase in platelet adhesion to angiogenic vessels of Matrigel compared with mature quiescent skin vessels. Our results suggest that the presence of platelets not only stimulates angiogenic vessel growth but also plays a critical role in preventing hemorrhage from the angiogenic vessels. The adhesion function of platelets, as mediated by GPIbalpha, significantly contributes to the process.

Animals↗
PubMed1996

Constitutive production of granulocyte/macrophage colony-stimulating factor by hypodense mononuclear eosinophils developed in vitro from hybrid eosinophil/basophil granulocytes.

We recently described the development in vitro of cells with granules characteristic of eosinophils and basophils (hybrid granulocytes) from normal human cord blood mononuclear cells cultured for 14 days with recombinant human (rh) interleukin (IL)-3, rhIL-5, and a soluble basement membrane, Matrigel. Hybrid granulocytes constitutively produced granulocyte/macrophage colony-stimulating factor (GM-CSF) and rapidly developed into eosinophils after the exogenous cytokines and Matrigel were removed. To characterize the developmental progression of hybrid granulocytes, cells were maintained for an additional 14 days in medium containing rhIL-3, rhIL-5, and Matrigel. After 28 days, 73% +/- 1% (mean +/- SEM; n = 6) of the nonadherent cells were mononuclear eosinophils, 13% +/- 3% were eosinophils with two or more nuclear lobes, 13% +/- 4% were hybrid granulocytes, and 0.2% +/- 0.1% were basophils. More than 90% of the mononuclear eosinophils were hypodense as determined by centrifugation through metrizamide gradients. After an additional 5 days of culture in medium without exogenous cytokines, 65% +/- 3% (n = 5) of the 28-day cells excluded trypan blue. In contrast, 2% +/- 1% of freshly isolated peripheral blood eosinophils survived 5 days of culture without exogenous cytokines (n = 5). Fifty percent conditioned medium from in vitro derived 28-day mononuclear eosinophils and 14-day hybrid granulocytes maintained the survival of 60% +/- 7% and 77% +/- 7%, respectively, of freshly isolated peripheral blood eosinophils for 72 h, compared with 20% +/- 8% survival in medium alone (n = 3). The eosinophil viability-sustaining activity of 50% mononuclear eosinophil-conditioned medium was neutralized with a GM-CSF antibody. A total of 88% of the 28-day cells exhibited immunochemical staining for GM-CSF. Thus, during eosinophilopoiesis, both hybrid eosinophil/basophil intermediates and immature mononuclear eosinophils exhibit autocrine regulation of viability due to constitutive production of GM-CSF.

Cell Survival↗
PubMed1998

De novo adipogenesis in mice at the site of injection of basement membrane and basic fibroblast growth factor.

Autografting of fat pads has a long history in plastic and reconstructive surgery for augmentation of lost soft tissue. However, the results are disappointing because of absorption of the grafts with time. The fate of transplanted fat is linked to adipose precursor cells distributed widely in connective tissues. Adipocyte precursor cells can proliferate and mature into adipocytes even in the adult body depending on microenvironment. When reconstituted basement membrane, Matrigel, supplemented with more than 1 ng/ml bFGF was injected s.c. into 6-week-old mice, the neovascularization induced within 1 week was followed by migration of endogenous adipose precursor cells, and a clearly visible fat pad was formed. The pad grew until 3 weeks after the injection and persisted for at least 10 weeks. Such de novo adipogenesis was induced reproducibly by s.c. injection of Matrigel and bFGF over the chest, lateral abdomen, or head. Adipogenesis could be induced even in ear cartilage or in muscle. Thus, our results demonstrated that an abundant population of adipose precursor cells is distributed widely in connective tissues of the adult body and that they migrate into the neovascularized plug of Matrigel for proliferation and maturation. These results suggest a technique of augmenting lost soft tissue in plastic and reconstructive surgery.

Adipocytes↗
PubMed1997

Misregulation of stromelysin-1 expression in mouse mammary tumor cells accompanies acquisition of stromelysin-1-dependent invasive properties.

Stromelysin-1 is a member of the metalloproteinase family of extracellular matrix-degrading enzymes that regulates tissue remodeling. We previously established a transgenic mouse model in which rat stromelysin-1 targeted to the mammary gland augmented expression of endogenous stromelysin-1, disrupted functional differentiation, and induced mammary tumors. A cell line generated from an adenocarcinoma in one of these animals and a previously described mammary tumor cell line generated in culture readily invaded both a reconstituted basement membrane and type I collagen gels, whereas a nonmalignant, functionally normal epithelial cell line did not. Invasion of Matrigel by tumor cells was largely abolished by metalloproteinase inhibitors, but not by inhibitors of other proteinase families. Inhibition experiments with antisense oligodeoxynucleotides revealed that Matrigel invasion of both cell lines was critically dependent on stromelysin-1 expression. Invasion of collagen, on the other hand, was reduced by only 40-50%. Stromelysin-1 was expressed in both malignant and nonmalignant cells grown on plastic substrata. Its expression was completely inhibited in nonmalignant cells, but up-regulated in tumor cells, in response to Matrigel. Thus misregulation of stromelysin-1 expression appears to be an important aspect of mammary tumor cell progression to an invasive phenotype.

Animals↗
PubMed2006

The novel SPARC family member SMOC-2 potentiates angiogenic growth factor activity.

SMOC-2 is a novel member of the SPARC family of matricellular proteins. The purpose of this study was to determine whether SMOC-2 can modulate angiogenic growth factor activity and angiogenesis. SMOC-2 was localized in the extracellular periphery of cultured human umbilical vein endothelial cells (HUVECs). Ectopically expressed SMOC-2 was also secreted into the tissue culture medium. In microarray profiling experiments, a recombinant SMOC-2 adenovirus induced the expression of transcripts required for cell cycle progression in HUVECs. Consistent with a growth-stimulatory role for SMOC-2, its overexpression stimulated DNA synthesis in a dose-dependent manner. Overexpressed SMOC-2 also synergized with vascular endothelial growth factor or with basic fibroblast growth factor to stimulate DNA synthesis. Ectopically expressed SMOC-2 stimulated formation of network-like structures as determined by in vitro matrigel angiogenesis assays. Fetal calf serum enhanced the stimulatory effect of overexpressed SMOC-2 in this assay. Conversely, small interference RNA directed toward SMOC-2 inhibited network formation and proliferation. The angiogenic activity of SMOC-2 was also examined in experimental mice by subdermal implantation of Matrigel plugs containing SMOC-2 adenovirus. SMOC-2 adenovirus induced a 3-fold increase in the number of cells invading Matrigel plugs when compared with a control adenoviral vector. Basic fibroblast growth factor and SMOC-2 elicited a synergistic effect on cell invasion. Taken together, our results demonstrate that SMOC-2 is a novel angiogenic factor that potentiates angiogenic effects of growth factors.

Adenoviridae↗
PubMed2006

NFAT induces breast cancer cell invasion by promoting the induction of cyclooxygenase-2.

The NFAT (nuclear factor of activated T cells) family of transcription factors plays a fundamental role in the transcriptional regulation of the immune response. However, NFATs are ubiquitously expressed, and recent evidence points to their important functions in human epithelial cells and carcinomas. Specifically, NFAT has been shown to be active in human breast and colon carcinoma cells and to promote their invasion through Matrigel. The mechanisms by which NFAT promotes invasion have not been defined. To identify NFAT target genes that induce carcinoma invasion, we have established stable breast cancer cell lines that inducibly express transcriptionally active NFAT. Gene expression profiling by cDNA microarray of cells induced to express NFAT revealed up-regulation of cyclooxygenase-2 (COX-2). Increased NFAT expression and activity induced COX-2 expression as well as prostaglandin E2 synthesis. This induction was more prominent when NFAT was activated by phorbol 12-myristate 13-acetate and calcium ionophore ionomycin and was blocked by the NFAT antagonist cyclosporin A. Breast cancer cells with elevated COX-2 expression showed increased invasion through Matrigel, and this was reduced in cells treated with COX-2 inhibitors. Conversely, loss of NFAT1 protein expression using small interfering RNA led to a reduction in COX-2 transcription and reduced invasion. Similarly, Matrigel invasion was reduced in cells in which COX-2 expression was reduced using specific siRNA. These findings demonstrate that NFAT promotes breast cancer cell invasion through the induction of COX-2 and the synthesis of prostaglandins.

Breast Neoplasms↗
PubMed2002

Active matrix metalloprotease-9 in and migration pattern of dendritic cells matured in clinical grade culture conditions.

Dendritic cells (DC) represent potent antigen presenting cells (APC) that are capable of generating tumor-specific immunity. In DC-based vaccination the migration of the infused DC from the site of injection to the secondary lymphoid organs might be critical to induce an effective immune response. Therefore we analyzed the migrating properties of maturing DC generated from human blood monocytes under culture conditions in compliance with the good manufacturing practice (GMP) guidelines. Highly purified CD14+ monocytes were differentiated into immature DC (iDC), then optimally matured as evidenced by CD83 expression. Time-lapse videomicroscopy and Transwell migration assays performed with or without Matrigel, proved mature DC (mDC) to be highly migrating cells compared to iDC although mDC migratory response varied markedly according to individuals (n= 15). Moreover, as shown by gelatin zymography and ELISA, mDC predominantly expressed both the active form of the matrix metalloprotease-9 (MMP-9) and low amounts of its physiological inhibitor, the tissue inhibitor of metalloprotease-1 (TIMP-1) which may explain their high migrating capacity through Matrigel layers. Macrophage inflammatory protein-3beta (MIP-3beta), strongly increased mDC migration through Matrigel by up-regulating the membrane MMP-9 active form suggesting that injected mDC could be selectively guided to T-cell areas of lymph nodes by this chemokine. Taken together, we demonstrate for the first time that mDC, but not iDC, prepared in clinical grade conditions are both physiologically invasive cells expressing chemokine-active and -sensitive MMP-9, which may be critical for their trafficking through tissues after injection. Consequently, we argue that migration characteristics should be included into a gold standard for DC administrated to patients.

Cell Differentiation↗
PubMed2006

In vivo and in vitro antitumor effect of a unique nutrient mixture on lung cancer cell line A-549.

The high incidence of lung cancer and ineffective toxic action of current mono and doublet chemotherapy approaches result in poor patient survival. Further, matrix metalloproteinases (MMPs) are implicated in neoplastic invasion and metastasis. Based on this, the authors investigated the effect of a dietary micronutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on the tumor growth of human lung carcinoma cell A-549 xenografts in athymic nude mice. Additionally, the authors tested the in vitro antitumor effect of NM on lung carcinoma A-549 cells by measuring cell proliferation by MTT assay, MMP-2 and -9 secretion by gelatinase zymography, and cell invasion through Matrigel. Nutrient supplementation strongly suppressed the growth of tumors without adverse effects in nude mice; tumor weight was reduced by 44% (P = .0001) and tumor burden was reduced by 47% (P < .0001) with supplementation. Zymography demonstrated in vitro secretion of MMP-2 by uninduced human lung carcinoma cells and both MMP-2 and -9 by phorbol 12-mysristate 13-acetate (PMA) (200 ng/mL)-treated cells. NM inhibited the secretion of both MMPs in a dose-dependent fashion, with virtual total inhibition at 500 microg/mL concentration. The invasion of human lung carcinoma cells through Matrigel was significantly reduced at 100 microg/mL (64%) and totally inhibited at 500 microg/mL concentration of NM (P = .01). Suppression of lung tumor growth in nude mice and inhibition of MMP secretion and Matrigel invasion suggest NM may act as an anticancer agent and as such warrants further investigation.

Adenocarcinoma↗
PubMed2001

Extracellular matrix-derived angiogenic factor(s) inhibit endothelial cell proliferation, enhance differentiation, and stimulate angiogenesis in vivo.

To isolate matrix molecules with angiogenic activity, tumor extracellular matrix (ECM) fractions from the basement membrane preparation Matrigel were analyzed for effects on endothelial cell (EC) proliferation, differentiation, and vessel formation in vivo. Inhibition of human and bovine EC DNA synthesis was evident upon treatment with several soluble Matrigel fractions including conditioned media (MGCM). After size fractionation of MGCM, EC growth arrest was activated by factor(s) smaller than 3,000 daltons (3KF). Bovine EC differentiation (tube formation) was promoted by both MGCM and 3KF fractions in two different models using matrigel or collagen gels to stimulate tube formation. The 3KF factor(s) stimulated angiogenesis when implanted in the cornea or subcutaneously in mice. FGF-induced angiogenesis and blood flow were increased in the presence of 3KF factor(s), an effect that was inhibited by the anti-angiogenic molecule endostatin. Further characterization of the low molecular weight 3KF samples by RP-HPLC revealed several fractions exhibiting EC growth arrest activity. These results suggest that the ability of ECM preparations to induce EC growth arrest and tube formation may reside, at least partially, in previously undetected low molecular weight molecules. Characterization of these ECM-associated inhibitors may lead to the development of novel anti-angiogenic and anti-tumor compounds.

Angiogenesis Inducing Agents↗
PubMed2005

Villous explant culture: characterization and evaluation of a model to study trophoblast invasion.

The mechanisms that control invasion of cytotrophoblast (CTB) cells into the maternal decidua and myometrium with transformation of the maternal spiral arteries are not fully understood, but oxygen is thought to be a key factor. We carried out a semiquantitative evaluation of an explant culture model for use in the study of trophoblast proliferation and invasion. Explants of human villous tissue (6-9 weeks of gestation) cultured on Matrigel in both standard culture conditions (18% O2) and in a low oxygen environment (2% O2) produced regions of outgrowth, of cytotrophoblast cells from villous tips and migration of cells into the Matrigel. The number of sites of outgrowth and migration, area of outgrowth, and extent of migration of cells into the Matrigel tended to increase throughout the culture period (144 h) but varied between explants from the same placenta and those from different placentas. There were no significant differences in the number of sites of outgrowth or migration scores in explants cultured in a low oxygen environment compared to those cultured in standard conditions. This study highlights the importance of careful validation, design and interpretation of experiments using in vitro culture systems, particularly those investigating the regulatory role of oxygen.

Abortion, Therapeutic↗
PubMed1995

In vivo angiogenesis induced by recombinant adenovirus vectors coding either for secreted or nonsecreted forms of acidic fibroblast growth factor.

In vivo gene transfer of angiogenic growth factors represents a potential approach to the treatment of ischemic diseases. The present study examined the in vitro and in vivo effects of two replication-deficient recombinant adenovirus (Ad) vectors coding for human acidic fibroblast growth factor (aFGF1-154). One vector codes for the nonsecreted form of the peptide (AdCMV.aFGF1-154), and the other vector codes for a recombinant, secreted form (AdCMV.sp+aFGF1-154). AdCMV.NLS beta gal, an adenovirus vector coding for beta-galactosidase (beta-Gal), was used as a control. Assessment of proliferation of starved human umbilical vein endothelial cells infected with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154 (20 pfu/cell) showed approximately 6- and 10-fold increase in cell number over control, respectively. Infection with AdCMV.sp+aFGF1-154 and with AdCMV.aFGF1-154 enhanced endothelial cell differentiation into capillary-like structures in vitro. However, this effect was significantly more pronounced with AdCMV.sp+aFGF1-154 than with AdCMV.aFGF1-154. Angiogenesis in vivo was assessed by injecting subcutaneously into mice 750 microliters of reconstituted basement membrane proteins (Matrigel) and the Ad vectors (2 x 10(8) pfu). After 14 days, there was histologic evidence of neovascularization in the animal's tissue surrounding the Matrigel plugs with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154. Further, the hemoglobin content of the Matrigel plugs with AdCMV.aFGF1-154 and with AdCMV.sp+aFGF1-154 was, respectively, 2.3- and 2.6-fold higher than with AdCMV.NLS beta gal. Together, these observations support the concept that adenovirus vectors coding for various forms of acidic FGF1-154 may be used to induce angiogenesis in vivo and may provide a new therapeutic approach to ischemic diseases.

Adenoviridae↗
PubMed1997

Urokinase and the urokinase receptor: association with in vitro invasiveness of human bladder cancer cell lines.

BACKGROUND AND PURPOSE: The plasminogen activators urokinase and tissue plasminogen activator are enzymes that degrade proteins in tissue basement membranes and the extracellular matrix (a biomolecular complex surrounding individual cells in tissues that serves as a barrier between the cells and the vascular and lymph systems). The action of these enzymes allows tumor cells to escape their local environment and metastasize. Plasminogen activator activity can be influenced by the urokinase receptor, which is expressed on the surface of cells, and by the plasminogen activator inhibitors 1 and 2. Because bladder tumors differ in their propensity to invade local areas and distant sites, we studied the expression of both plasminogen activators, the two plasminogen activator inhibitors, and the urokinase receptor in four human bladder cancer cell lines (RT4, 253J, EJ, and T24) to see if there was an association between the expression of these proteins and tumor cell invasiveness in vitro. METHODS: The expression of urokinase, tissue plasminogen activator, and the two inhibitors was measured by enzyme-linked immunosorbent assays of serum-free, cell-conditioned media (i.e., culture fluids). Cell surface expression of the urokinase receptor was assayed by flow cytometry, using an anti-receptor monoclonal antibody (Mab3936). The invasive capacity of untreated cells and of cells exposed to exogenous, high-molecular-weight urokinase was analyzed by use of Matrigel invasion chambers. RESULTS: The four bladder cancer cell lines demonstrated differential expression of both plasminogen activators and both inhibitors; three of the cell lines (T24, EJ, and 253J) expressed the urokinase receptor. The four cell lines differed in their invasive potential in vitro. Neither expression of tissue plasminogen activator nor production of the inhibitors appeared to influence Matrigel invasion. EJ cells and 253J cells produced the highest levels of urokinase and demonstrated the greatest propensity for invasion; T24 cells, which produced only small amounts of urokinase, exhibited a low invasive potential. Pretreatment of T24 cells with exogenous high-molecular-weight urokinase markedly increased their invasiveness. Similar pretreatment of EJ and 253J cells increased their invasiveness as well. RT4 cells, which lacked urokinase receptor expression but produced moderate amounts of urokinase, were not invasive and did not become so after exposure to exogenous high-molecular-weight urokinase. Binding of Mab3936 to urokinase receptors inhibited Matrigel invasion. CONCLUSIONS: To our knowledge, this is the first study demonstrating that bladder tumor cells express the urokinase receptor and that both receptor expression and urokinase expression are required for bladder tumor cell invasion in vitro.

Carcinoma, Transitional Cell↗
PubMed1998

Colorectal carcinoma invasion inhibition by CO17-1A/GA733 antigen and its murine homologue.

BACKGROUND: The gastrointestinal carcinoma antigen GA733 is a potential target for passive and active immunotherapy for patients with colorectal carcinoma. This antigen has been characterized previously as a homophilic adhesion (i.e., adhesion to self) protein, but the functional consequences of homophilic adhesion for tumor growth and invasion are unknown. The availability of a murine homologue of GA733, i.e., murine epithelial glycoprotein (mEGP), allows for functional analysis of cell adhesion as it relates to tumor growth and invasion, both in vitro and in vivo. METHODS: CT-26 murine colorectal carcinoma cells were transfected with complementary DNAs encoding either the human or the murine antigen. GA733- or mEGP-producing cells were evaluated for homophilic adhesion, growth on plastic surfaces, colony formation in soft agar, and invasion through a reconstructed basement membrane (Matrigel). mEGP-producing cells were also examined for their capacity to metastasize in mice. Reported P values are two-sided. RESULTS: Compared with control cells, mEGP-producing cells showed significantly lower growth rates, colony formation, and invasion through Matrigel in vitro (all P values <.05). Compared with vector-only transfected cells and parental cells, mEGP-producing cells showed a reduction in metastatic potential in syngeneic immunodeficient and immunocompetent mice (all P values <.05). In contrast to mEGP-transfected cells, GA733-transfected cells did not exhibit significantly reduced growth or colony formation in vitro (all P values >.05). However, GA733-transfected cells did show reduced invasion through Matrigel compared with vector-only transfected cells or parental cells (all P values <.05). CONCLUSION: The adhesion proteins GA733 and mEGP inhibit invasion of tumor cells.

Animals↗
PubMed2004

Mesangial cells stimulate differentiation of endothelial cells to form capillary-like networks in a three-dimensional culture system.

BACKGROUND: There are conflicting results regarding the role of periendothelial mural cells in angiogenesis. In the current study, we investigated the role of mesangial cells (MCs) in endothelial vascularization by using a three-dimensional co-culture system in basement-membrane reconstruct gel (Matrigel). METHODS: Human umbilical vein endothelial cells (ECs) and human MCs were used. In the contact co-culture system, ECs and MCs were mixed and then plated together onto Matrigel. In the non-contact co-culture system, MCs were cultured within an intercup chamber, which prevented direct physical contact with the ECs on the Matrigel but allowed both cell types to share culture medium. To visualize ECs and MCs, the cells were labelled with two different fluorescent dyes prior to the co-culture. A capillary-like network formation was observed under a fluorescent microscope and confocal microscope, and the length of the network formation was quantified by the image analyzer. RESULTS: ECs barely formed capillary-like networks when cultured alone in growth factor-free medium. However, ECs cultured with MCs in a contact condition remarkably formed capillary-like networks (9.10+/-0.96 vs 0.20+/-0.07 mm/mm2 at 6 h, contact vs ECs alone, P<0.001). Direct contact between ECs and MCs was clearly demonstrated by confocal microscopy. Differentiation into branching capillary-like structures was also observed in the non-contact co-culture system (3.02+/-1.21 mm/mm2 at 6 h, P<0.001 vs ECs alone), but less prominently than in the contact co-culture condition. Vascular endothelial growth factor (VEGF) was secreted from MCs, as determined by enzyme-linked immunosorbent assay and immunofluorescent study. Adding neutralizing antibodies against VEGF into the co-culture system partially inhibited capillary network formation. CONCLUSIONS: Our data indicate that MCs help ECs differentiate toward vascularization, in which the direct cell-cell contact between ECs and MCs plays an important role. VEGF is a mediator in this process.

Analysis of Variance↗