Naoki Utoguchi

Nitric Oxide Donors Enhance Rectal Absorption of Macromolecules in Rabbits

AbstractPurpose. The objective of this investigation is to evaluate the potential of nitric oxide (NO) donors as a new class of absorption enhancers which may act on intestinal epithelial cells through epithelial actions of the chemical mediator, NO. Methods. Suppositories containing NO donors and insulin were administered into the rabbit rectum. After administration of the suppository, blood samples were collected from the auricular vein. The plasma insulin and glucose concentrations were determined. Results. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP, 4 mg) induced a significant increase in the rate of insulin absorption from the rectum. Administration of a suppository containing SNAP without insulin affected neither the plasma insulin nor the plasma glucose concentration. Other NO donors, NOR1 and NOR4, also induced increases in the insulin absorption. The absorption enhancement effect of SNAP was inhibited by coadministration of the NO scavenger carboxy-PTIO. SNAP also enhanced FITC-dextran (MW 4,000) absorption. Little cytotoxicity of SNAP (3.0 mg/ml) as assessed in terms of the rate of lactate dehydrogenase (LDH) release from Caco-2 cells was detected for 2 h of incubation. Conclusions. These findings suggest that NO enhanced macromolecular absorption from the rectum without mucosal cell damage, and that NO donors can act as potent absorption enhancers.

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.

Effect of tumour cell-conditioned medium on endothelial macromolecular permeability and its correlation with collagen

Conditioned medium prepared from mouse melanoma B16 cells (B16-CM) increases the macromolecular permeability of bovine aortic, venous and human umbilical vein endothelial monolayer. Collagen, which is synthesised by endothelial cells, has an important function in regulating the permeability of endothelial monolayer. Briefly, low collagen content leads to hyperpermeable structure of the endothelial monolayer. In the present studies, we examined the relationship between the increase of endothelial permeability and content of synthesised collagen of endothelial cells cultured with B16-CM. The B16-CM reduced endothelial collagen content but did not digest collagen directly. Matrix metalloproteinase inhibitor, 1,10-phenanthroline, inhibited the increase in permeability due to addition of B16-CM. These data suggest that B16-CM acts on endothelial cells, stimulating the digestion of endothelial collagen, and that the reduced content of collagen leads to the hyperpermeability of the endothelial monolayer.

Tumor-conditioned medium increases macromolecular permeability of endothelial cell monolayer

The permeation of macromolecular FITC-labeled dextran (molecular weight 70,000) through bovine aortic endothelial cells (BAEC) monolayer, which were cultured for 5 days with conditioned medium prepared from mouse melanoma B16, was increased. However, when BAEC, which were cultured with normal medium until confluent, were treated with B16 conditioned medium (B16-CM) for 30 min, the permeability did not increase. The B16-CM also increased the permeability of the endothelial monolayers of bovine veins and the human umbilical vein, but did not increase that of the epithelial monolayer. The B16-CM did not alter the distribution or content of F-actin on the BAEC. BAEC cultured in the presence of B16-CM for 5 days were detached from the dish, and then seeded into a chamber at one-fifth of confluent cell density. After 5 days of culture in normal medium, the BAEC were grown to confluence and their permeability was increased. These findings suggest that B16-CM increased the endothelial permeability irreversibly without the decrease of F-actin, and that soluble factor(s) which were secreted from the tumor cells participate in the construction of the hyperpermeable structure of tumor vessels in vivo.

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.

Effective Cancer Targeting Using an Anti‐tumor Tissue Vascular Endotheliumspecific Monoclonal Antibody (TES‐23)

Immunoconjugate targeting of solid tumors has not been routinely successful because the endothelial 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 antitumor 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 125I‐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.

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.

SummaryThe 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.

Glial extracellular matrix modulates γ-glutamyl transpeptidase activity in cultured bovine brain capillary and bovine aortic endothelial cells

Glial extracellular matrix (ECM) elevated gamma-glutamyl transpeptidase (gamma-GTP) activity in cultured bovine brain capillary and aortic endothelial cells (BBCEC, BAEC). In particular, the ECM of glial cells cultured with the conditioned medium of BAEC (BAEC CM) dramatically elevated gamma-GTP activity in BBCEC and BAEC. The ECM of glial cells cultured with BBCEC CM also had a marked effect. The ECM of 3T3 cells cultured with BAEC CM, and the ECM of glial cells cultured with 3T3 CM had no effect. Glial CM had no effect on gamma-GTP activity in BBCEC and BAEC. These findings indicate that gamma-GTP activity in endothelial cells (EC) is modulated by glial ECM, and that the factor of ECM that affects gamma-GTP activity in EC arises from the interaction between glial cells and EC.

Isolation and Properties of Tumor-derived Endothelial Cells from Rat KMT-17 Fibrosarcoma

Rat KMT‐17 fibrosarcoma‐derived endothelial cells were isolated by Percoll gradient centrifugation with an attaching‐speed separation technique, and their properties in culture were examined. The primary cultured tumor‐derived endothelial cells (TEC) showed angiotensin‐converting enzyme activity, positivity for Factor VIII‐related antigen staining, and typical capillary‐like formation on Matrigel. The primary cultured TEC monolayer showed greater permeability than normal tissuederived endothelial cell (aorta, vena cava and epididymal fat capillary) monolayers on FITC‐dextran diffusion (molecular weight 70,000). Leukocyte adhesion to TEC was reduced compared to that to fat‐derived capillary endothelial cells. These characteristics resembled those of tumor vascular endothelium, and were observed both in the primary and first‐passage cell cultures, but not in the fourth‐passage cell cultures. Our findings indicate that primary or subcultured TEC are applicable for studies of the physiological characteristics of tumor endothelial cells.

Improvement of Intestinal Absorption of Macromolecules by Nitric Oxide Donor

Nitric oxide (NO) is one of the most versatile mediators in mammalian biology. In the present study, we investigated the absorption-enhancing effects of an NO donor, 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propa namine (NOC7), on drugs that are poorly absorbed from the gastrointestinal tract. NOC7 significantly increased the jejunal absorption of fluorescein isothiocyanate dextrans (FDs) of different average molecular weights (4000-20,000). This enhancing effect decreased as the FD molecular weight increased. Another NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), also increased the absorption of FD-4 from the jejunum. The absorption enhancement effect of NOC7 significantly decreased after coadministration with an NO scavenger, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazole-1-oxyl-3-oxide, sodium salt. Furthermore, the enhancement effect of NOC7 was reversed shortly after cessation of the enhancer treatment. Little damage by NOC7 to the intestinal mucosa was observed in terms of release of lactose dehydrogenase and protein from the intestinal mucosa. NOC7 also increased the absorption of FD-4 by the colon and rectum. The findings suggest that an NO donor can improve the absorption of macromolecules from all regions of the rat intestine with very little mucosal damage and that an NO donor can act as a potent absorption enhancer.