Efflux transporter mRNA expression profiles in differentiating JEG-3 human choriocarcinoma cells as a placental transport model.

The kinetics of drug transport across the trophoblast layer is determined by several factors. Human choriocarcinoma cell lines like BeWo and JEG-3 have been used as models of the trophoblast layer to examine the placental transport of drugs. Previously, the drugs examined in these models have been readily transported across the trophoblast layer via cellular gap junctions. These backgrounds enabled us to establish the differentiating JEG-3 cell (DJEG) layer model, which suppresses paracellular drug transport, as an evaluation system of placental drug transport. The efflux transporters on the trophoblast layer assume the meaningful role of protecting the fetus from xenobiotic substances. In order to clarify the usefulness of our DJEG placental drug transport model, this study examined the mRNA expression profiles of the efflux transporters MRPs, MDR1, and BCRP in JEG-3 cells and compared them with those of BeWo cells and their known placental expression. We suggest that the mRNA of efflux transporters MRP 1-8 and BCRP are expressed widely in JEG-3 cells; however, expression levels of MDR1 mRNA were undetectable. It was also indicated that polymorphisms of BCRP C421A in both the BeWo and JEG-3 cells are of the wild-type. We demonstrated the efflux transporters' expression profiles, as well as those of the BeWo cells, was demonstrated in the DJEG placental drug transport evaluating model as well as the BeWo cells, in the DJEG placental drug transport evaluation model. Based on these findings, we hope that the DJEG model will be adequate for use in evaluating placental drug transport in relation to the transporter proteins.

Effect of forskolin on the expression of claudin-5 in human trophoblast BeWo cells.

Trophoblasts, a cell type found in the placenta, play a pivotal role in the function of the placenta as a barrier between the maternal fluid and the fetus. Recently, claudin, a 24-kDa transmembrane protein, was identified as being responsible for the barrier function of epithelia. In the present study, we investigated the expression profiles of claudin and the changes in expression during the differentiation of BeWo human trophoblast cells. Reverse transcriptase-polymerase chain reaction and immunoblotting demonstrated the expression of claudin-1, -3, -4, and -5 in BeWo cells. Forskolin, which induces the differentiation of BeWo cells from cytotrophoblast-like cells into syncytiotrophoblast-like cells, reduced slightly the expression of claudin-5. This is the first report to show changes in claudin-5 in forskolin-treated BeWo cells.

Characteristics of transcription-regulatory elements for gene expression from plasmid vectors in human trophoblast cell lines.

Nonviral gene delivery systems are useful for basic research in trophoblasts. In these systems, gene expression is regulated by a cassette of regulatory elements within the plasmid, and the transcriptional activity differs among cell lines. In the present study, we used BeWo and JAR human trophoblast cell lines to systematically compare the transcriptional activities of several expression cassettes and those of a control plasmid made up of a simian virus 40 (SV40) promoter, a polyadenylation (PA) signal, and an enhancer. We also found that insertion of intron elements enhanced transcriptional activities in the following order: intron A>hybrid beta-globin-immunoglobin intron>no intron. Of several PA signals tested including those from SV40, bovine growth hormone, and the minimal rabbit beta-globin, the latter had the highest transcriptional activities (3.9- and 26-fold over control plasmid in BeWo and JAR cells, respectively). Addition of a second enhancer increased the transcriptional activity in these cells. We also found that gene expression level can be controlled by selecting the expression cassette. These results should be useful for further transgene experiments in BeWo and JAR cells.

CAR- or alphav integrin-binding ablated adenovirus vectors, but not fiber-modified vectors containing RGD peptide, do not change the systemic gene transfer properties in mice.

Targeted gene delivery to the tissue of interest by recombinant adenovirus (Ad) vectors is limited by the relatively broad expression of the primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, alphav integrin. This problem could be overcome by mutating the fiber and penton base, which bind with CAR and alphav integrin, respectively. In this study, we constructed CAR-binding ablated Ad vectors and alphav integrin-binding ablated Ad vectors by mutation in the FG loop of fiber knob and in the RGD motif of penton base, respectively, and compared the gene transfer properties of their vectors into various types of cultured cells and mice with conventional Ad vectors. We also generated Ad vectors containing RGD peptide in the HI loop of the fiber knob. CAR-binding ablated Ad vectors mediated about 1% of gene transfer activity into CAR-positive cultured cells, compared with conventional Ad vectors, while alphav integrin-binding ablated Ad vectors maintained at least 76% of gene transfer activity into cultured CAR-positive cells. Inclusion of the RGD peptide into the HI loop of the fiber knob of CAR-binding ablated Ad vectors restored gene transfer activity in vitro. On the other hand, systemically administered CAR-binding ablated Ad vectors, as well as alphav integrin-binding ablated Ad vectors mediated similar levels of gene transfer into mouse liver with the conventional Ad vectors. These results suggest that continued interaction of either the fiber with CAR or the penton base with alphav integrin offers an effective route of virus entry into mouse liver in vivo. Inhibition of the interaction of both the fiber with CAR and the penton base with alphav integrin is likely to be crucial to the development of targeted Ad vectors.

Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide.

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed alpha(v) integrin expression, about 10-1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (alpha(v)beta3 and alpha(v)beta5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.

Carrier-mediated transport of folic acid in BeWo cell monolayers as a model of the human trophoblast.

Using cultured BeWo cells as a model of human trophoblast, we investigated whether carrier-mediated transport of folic acid occurs. BeWo cells, which were derived from human choriocarcinoma, were cultured on a tissue culture plate or in a permeation chamber. When the cells reached confluence, drug uptake or transport experiments were performed. The uptake of [(3)H]folic acid by BeWo cells occurred at a much lower rate at 4 degrees C than at 37 degrees C. The uptake of [(3)H]folic acid was saturable at higher concentrations and inhibited by typical metabolic inhibitors, sodium azide and 2,4-dinitrophenol. The uptake of [(3)H]folic acid was significantly increased with decreasing pH of the incubation buffer and markedly inhibited by 4,4'-diidothiocyanostilbene-2,2'-disulfonic acid (DIDS). Analogs of folic acid, methotrexate and 5-methyltetrahydrofolate, inhibited the uptake of [(3)H]folic acid by BeWo cells. Kinetic analysis using Lineweaver-Burk plots revealed that methotrexate competitively inhibited the uptake of [(3)H]folic acid and folic acid competitively inhibited the uptake of [(3)H]methotrexate. In transport experiments, the permeation of [(3)H]folic acid from the apical-to-basal side was greater than that from the basal-to-apical side, and the transport of [(3)H]folic acid from the apical-to-basal side was inhibited by an excess of folic acid. The findings obtained in the present study confirm the existence of an asymmetric, carrier-mediated transport system for folic acid and its analog, methotrexate, across BeWo cells, a representative of the human trophoblast.

A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob.

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.

Effective cancer targeting using an anti-tumor tissue vascular endothelium-specific monoclonal antibody (TES-23).

Immunoconjugate targeting of solid tumors has not been routinely successful because the endo-thelial cells of blood vessels act as a physical barrier against the transport of macromolecules, such as antibodies. In the present study, we attempted to achieve tumor vascular targeting with an anti-tumor tissue endothelium-specific monoclonal antibody (TES-23). TES-23, an IgG1 monoclonal antibody raised against rat KMT-17 fibrosarcoma-derived endothelial cells, was covalently conjugated with neocarzinostatin (NCS) in a previous study. The TES-23-NCS conjugate induced tumor hemorrhagic necrosis, and showed marked anti-tumor effects against rat KMT-17 fibrosarcoma. This result prompted us to investigate whether this approach would be applicable to various other types of solid tumors. One hour after injection of (125)I-labeled TES-23 into BALB / c mice bearing Meth-A fibrosarcoma and Colon 26 adenocarcinoma, the tumor accumulation of TES-23 was greater than that of the control IgG. In the present study, we report the anti-tumor effects of this monoclonal antibody in mice bearing Meth-A fibrosarcoma. Mice treated with the immunoconjugate showed improved survival with no side effects. This result indicates that common antigens may be found in different kinds of tumor endothelial cells, and that TES-23 might recognize these antigens.

Absorption enhancement of a protein drug by nitric oxide donor: effect on nasal absorption of human granulocyte colony-stimulating factor.

The objective of this study was to evaluate the effect of a nitric oxide (NO) donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the nasal absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rabbits and to evaluate the irritation (cytotoxicity) potential of the NO donor on the mucosal membrane using a cultured cell system (strain KB, human epidermoid carcinoma of the floor of the mouth). Significantly higher serum G-CSF concentration and increased total leukocyte count in the peripheral blood were observed after coadministration of rhG-CSF (100 microg/kg) with SNAP at various doses (0.3-3.3 mg/kg). The serum G-CSF concentration and the increased total leukocyte count were markedly decreased by the presence of the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazole-1-oxyl 3-oxide sodium salt (carboxy-PTIO), in combination with rhG-CSF and SNAP. However, no significant inhibitory effect of glutathione (peroxynitrite scavenger) on the absorption-enhancing effect of SNAP was observed. These results suggest that carboxy-PTIO inhibits the absorption-enhancing effect of NO released from SNAP. We found that SNAP has a very low potential for cytotoxicity, as evaluated by the cell detachment assay, release of lactate dehydrogenase (LDH) from cultured cells and morphological observations of nasal tissue of rabbits. It is concluded that a NO donor such as SNAP is a promising absorption enhancer for nasal protein-drug delivery.

Functional expression of P-glycoprotein in primary cultures of human cytotrophoblasts and BeWo cells.

The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers. Uptake studies with primary cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors. Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole. MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells. Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells. This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta.

Carrier-mediated transport of valproic acid in BeWo cells, a human trophoblast cell line.

The biochemical mechanisms mediating the rapid distribution of valproic acid across placenta are not precisely known. We have characterized valproic acid transport by the human trophoblast using the human choriocarcinoma cell line, BeWo. The uptake of [14C]valproic acid by BeWo cells was found to be saturable and blocked by pre-exposure to the metabolic inhibitors, sodium azide and 2,4-dinitrophenol. Valproic acid uptake by the BeWo cells was also inhibited by the protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, but not anion exchange inhibitor. Selected monocarboxylic acids inhibited the uptake of [14C]valproic acid by BeWo cells, whereas dicarboxylic acids did not alter the uptake process. Analysis of Lineweaver-Burk plots of valproic acid uptake in the presence of benzoic acid, a marker for the monocarboxylic acid transporter, revealed a competitive process for uptake. In transcellular transport experiments, the permeation of [14C]valproic acid from the apical-to-basal side of the monolayers was significantly greater than the permeation from basal-to-apical side. Additionally, the permeation of [14C]valproic acid from apical-to-basal side was inhibited by monocarboxylic acids and not dicarboxylic acids. The results provide biochemical evidence of a proton-dependent, saturable, and asymmetric transport system, presumed to be a monocarboxylic acid transporter, for valproic acid in a human trophoblast model.

Specific binding of TES-23 antibody to tumour vascular endothelium in mice, rats and human cancer tissue: a novel drug carrier for cancer targeting therapy.

The tissue distribution of anti-tumour vascular endothelium monoclonal antibody (TES-23) produced by immunizing with plasma membrane vesicles from isolated rat tumour-derived endothelial cells (TECs) was assessed in various tumour-bearing animals. Radiolabelled TES-23 dramatically accumulated in KMT-17 fibrosarcoma, the source of isolated TECs after intravenous injection. In Meth-A fibrosarcoma, Colon-26 adenocarcinoma in BALB/c mice and HT-1080 human tumour tissue in nude mice, radioactivities of 125I-labelled TES-23 were also up to 50 times higher than those of control antibody with little distribution to normal tissues. The selective recognition of TES-23 to TECs was competitively blocked by preadministration of unlabelled TES-23 in vivo. Furthermore, immunostaining of human tissue sections showed specific binding of TES-23 on endothelium in oesophagus cancers. These results indicate that tumour vascular endothelial cells express common antigen in different tumour types of various animal species. In order to clarify the efficacy of TES-23 as a drug carrier, an immunoconjugate, composed of TES-23 and neocarzinostatin, was tested for its anti-tumour effect in rats bearing KMT-17 fibrosarcomas. The immunoconjugate (TES-23-NCS) caused marked regression of the tumour, accompanied by haemorrhagic necrosis. Thus, from a clinical view, TES-23 would be a novel drug carrier because of its high specificity to tumour vascular endothelium and its application to many types of cancer.

Carrier-mediated transport of monocarboxylic acids in BeWo cell monolayers as a model of the human trophoblast.

The monolayer-forming, human choriocarcinoma cell line, BeWo, was used to study the mechanisms of monocarboxylic acid transport across the human trophoblast. Benzoic acid, acetic acid, and lactic acid were used as markers for monocarboxylic acid carrier-mediated transport. The uptake of benzoic acid by BeWo cells was saturable (K(t) = 0.6 +/- 0.3 mM) at higher concentrations and significantly inhibited by typical metabolic inhibitors, sodium azide and 2, 4-dinitrophenol. A selection of different monocarboxylic acids, including a natural substrate lactic acid, also substantially inhibited the uptake of benzoic acid and acetic acid by BeWo cells, whereas dicarboxylic acids did not affect the uptake of either marker. Monocarboxylic acid uptake was pH-dependent and inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore. Kinetic analysis using Lineweaver-Burk plots revealed that monocarboxylic acids competitively inhibited the uptake of benzoic, lactic, and acetic acid by BeWo cells. In transport experiments, the permeation of benzoic acid from apical-to-basolateral side was greater than the permeation from the basolateral-to-apical side, and the transport of benzoic acid from apical-to-basolateral side was inhibited by monocarboxylic acids. The findings obtained in the present study confirm the existence of an asymmetric, carrier-mediated transport system for monocarboxylic acids across the BeWo cell, a representative of the human trophoblast.

Suppression of solid tumor growth by a monoclonal antibody against tumor vasculature in rats: involvement of intravascular thrombosis and fibrinogenesis.

We have reported that immunization of rat tumor-derived endothelial cells (TEC) isolated from KMT-17 solid tumors results in the generation of several monoclonal antibodies (MAbs). TES-23, one of these MAbs, recognizes a naturally occurring 80-kDa antigen expressed on endothelial cells of tumor blood vessels. To determine whether such MAbs can suppress solid tumor growth in vivo by impairment of endothelial cells in tumors following direct binding, we tested the biodistribution of (125)I-labeled TES-23 in rats bearing KMT-17 solid tumors. We also examined the effect of treatment using unconjugated TES-23 on tumor growth and histo-pathological changes in tumor tissues. Biodistribution studies showed localization of TES-23 into tumor tissues 60 min after intravenous injection. TES-23 suppressed significantly the growth of KMT-17 solid tumors following administration for 5 days. Histo-pathological examination showed that TES-23 caused degeneration, apoptosis and/or necrosis and denudation of endothelial cells in viable tumor areas following local aggregation and adhesion of lymphocytes, with subsequent intravascular thrombus formation by platelets and fibrin. Our results indicate that TES-23, which recognizes TEC, can target endothelial cells of solid tumor vasculature directly, resulting in growth suppression in vivo by reduction of blood flow due to intravascular thrombosis. Our results also suggest that targeting tumor vasculature is a potentially attractive approach for the treatment of solid tumors.

Tumor vascular targeting using a tumor-tissue endothelium-specific monoclonal antibody as an effective strategy for cancer chemotherapy.

In this study, we attempted to develop tumor vascular targeting with a tumor tissue endothelium-specific monoclonal antibody. TES-23, which strongly and selectively recognizes tumor tissue endothelial cells, was chemically conjugated with Neocarzinostatin (NCS), and the anti-tumor effect was examined. The immunoconjugate, TES-23-NCS, showed, through the use of tumor hemorrhagic necrosis, a marked anti-tumor effect on KMT-17 tumors in rats at a dosage of 17 micrograms/kg (NCS equivalent) without any side effects, probably due to specific tumor vascular injury. By contrast, TES-23 alone (107 micrograms/kg), NCS alone (17 micrograms/kg), and Mopc-NCS (Mopc, 107 micrograms/kg; NCS, 17 micrograms/kg), the immunoconjugate of control antibody, did not have any anti-tumor activities. By tissue distribution analysis, TES-23 and TES-23-NCS showed high accumulation in KMT-17 tumors 1 h after intravenous administration. Moreover TES-23 also accumulated in Sarcoma-180 tumors in mice 1 h after intravenous administration. These results suggest that TES-23 may be a candidate for a potential tumor vascular targeting agent that is applicable to a wide variety of tumor types.

Different reactions of aortic and venular endothelial cell monolayers to histamine on macromolecular permeability: role of cAMP, cytosolic Ca2+ and F-actin.

Endothelial cells assume a central role in the one process that the permeation of microvessels is accelerated in case of inflammation. We studied the effect of histamine on endothelial permeability, [Ca2+]i, cAMP and F-actin, using same origin aortic and venular cultured endothelial monolayers. When HUVEC were treated with histamine (10(-7)-10(-5) M), permeability of FITC-dextran (molecular weight 70,000) and [Ca2+]i were increased, while cAMP content was unchanged, and F-actin content was reduced. When bovine vein-derived endothelial cells were treated with histamine, [Ca2+]i was increased via H1 receptors, but permeability and F-actin content were not altered. When human aorta-derived endothelial cells were, [Ca2+]i was increased via H1 receptors and cAMP content was increased via H2 receptors, while permeability and F-actin content were not changed. When bovine aorta-derived endothelial cells were, cAMP and F-actin content were increased, while permeability was reduced. These findings suggest that endothelial cells derived from different tissues clearly showed the different reactions to histamine, the increase in [Ca2+]i led to the increase in endothelial permeability, while the increase in cAMP levels led to the reduction in permeability, and finally, F-actin regulated endothelial macromolecular permeability.

Carrier-mediated absorption of salicylic acid from hamster cheek pouch mucosa.

Previously, we found that monocarboxylic acids undergo carrier-mediated transport in primary cultures of oral mucosal epithelial cells.1 In this study, we investigated whether carrier-mediated absorption of a monocarboxylic acid from the oral mucosa occurs in vivo. Salicylic acid was administered to hamster cheek pouch. At predetermined intervals, the concentration of salicylic acid in the fluid remaining in the cheek pouch lumen and the blood salicylic acid concentration were determined. The absorption of salicylic acid was saturable at high salicylic acid concentrations. Sodium azide, a metabolic inhibitor, and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore, significantly inhibited the absorption of salicylic acid but not the absorption of salicylamide from the oral mucosa. Various monocarboxylic acids inhibited the absorption of salicylic acid, whereas dicarboxylic acids had no such effect. Transfer of [14C]salicylic acid from the cheek pouch mucosa to the systemic circulation was observed, and the blood [14C]salicylic acid concentration in the case of coadministration with propionic acid was significantly lower than that in the case of no propionic acid coadministration. These results show that monocarboxylic acids undergo carrier-mediated absorption from the hamster cheek pouch mucosa.

Drug resistance and P-glycoprotein expression in endothelial cells of newly formed capillaries induced by tumors.

The inhibitory effects of vincristine (VCR) with or without verapamil (VER) on angiogenesis in mouse subcutaneous fascia induced by mouse sarcoma 180 cells were assessed using the dorsal air sac method. VCR combined with VER had inhibitory effects on tumor-induced angiogenesis, but VCR alone did not inhibit capillarization. The chemosensitivity of human umbilical vein endothelial cells (HUVEC) and tumor-derived endothelial cells from rat KMT-17 fibrosarcoma (TEC) was examined using the microculture tetrazolium assay. VCR and taxol had strong anti-proliferative activity against HUVEC, but only weakly inhibited the proliferation of TEC. In combination with VER, VCR and Taxol inhibited the proliferation of TEC. Cisplatin, mitomycin C, and 5-fluorouracil had weakly anti-proliferative activity against both HUVEC and TEC. Expression of P-glycoprotein (P-gp) was found in TEC, but not in HUVEC using western blot analysis. These findings indicate that drug resistance and P-gp expression appeared on newly formed capillaries induced by rodents tumors.

Identification of tumor vascular antigens by monoclonal antibodies prepared from rat-tumor-derived endothelial cells.

We have reported the isolation and specific in vitro properties of tumor-derived endothelial cells (TEC) from rat KMT-17 fibrosarcomas transplanted into rats. To develop antibody-based tumor vascular targeting therapy for solid tumors, we have generated monoclonal antibodies (MAbs) using passive immunization of outside-out membrane vesicles of rat epididymal-fat-pad-derived capillary endothelial cells (FCEC) followed by active immunization of those of rat TEC. The MAbs produced were screened against TEC and FCEC. Of all cultured hybridomas, 75 (3.3%) of the secreted MAbs preferentially recognized TEC. We selected a total of 7 MAbs which detected antigens highly abundant in TEC, although 5 of the 7 MAbs were weakly positive for FCEC in cell-ELISA and FACS analyses. The antigens recognized by these MAbs, with the exception of MAb TES-7, were present on endothelial cells of tumor blood vessels in KMT-17 fibrosarcoma tissues, as shown by immunohistochemical analysis. Antigens of 40- and 80-kDa were recognized by MAbs TES-1, 7, 17, 21 and 26 and by MAbs TES-23 and 27 respectively. Although the function of these antigens, which are preferentially expressed on rat tumor-derived endothelial cells, is still unknown, we believe that future studies of such antigens will help elucidate the role of endothelial cells in tumor vasculature. Our results indicate that MAbs may provide a novel tool for the development of antibody-based therapy targeting tumor vasculature.

Preparation of glial extracellular matrix: a novel method to analyze glial-endothelial cell interaction.

Studies on the interactions of endothelial cells and glial cells are of increasing importance for the understanding of the formation of the blood-brain barrier (BBB) and for the reconstruction of BBB properties in cultured brain capillary endothelial cells in vitro. Many methods have been used to examine cell-cell interactions, including conditioned medium, co-culture, feeder layers, and many others. Here we describe how to prepare the extracellular matrix (ECM) secreted from cultured cells. Cells are known to produce and interact with their extracellular components in an organized matrix and to regulate the function of other cells through the ECM. The ECM plays a central role in the differentiation and function of the cells, and controls the proliferation and motility of these cells. The responses of cells to ECM molecules need to be clarified. As the ECM is situated between cerebral capillaries and astrocytes in the central nervous system, the ECM secreted by glial cells may also play an important role in the formation and maintenance of the BBB. In our previous studies, the ECM produced by glial cells elevated gamma-glutamyl transpeptidase activity, which is an accepted marker enzyme for differentiated brain capillary endothelial cells, in cultured bovine brain capillary and aortic endothelial cells. Using the method described here, the cell-cell interaction via the ECM molecules can be examined.