竹内 公一

Background/Aim: Gene transfer into the kidney has great potential as a novel therapeutic approach. However, an efficient method of gene transfer into the kidney has not been established. We explored the transduction efficiency of renal cells in vitro and in vivo using adeno-associated virus (AAV) serotype 1-5 vectors encoding the β-galactosidase gene. Methods: In the in vitro study, rat kidney epithelial cell line NRK52E cells were transfected with AAV serotype derived vectors. In the in vivo study, AAV serotype derived vectors were selectively injected into the kidney using a catheter-based gene delivery system in rats and mice mimicking the clinical procedure. The efficiency of gene expression was histologically evaluated on the basis of the β-galactosidase expression. Results: AAV serotype 1, 2, and 5 vectors transduced in rat kidney epithelial cell line NRK52E cells in vitro, whereas AAV serotype 3 or 4 vectors showed no transduction. In addition, the kidney-specific injection of AAV serotype 2 vectors successfully transduced in tubular epithelial cells, but not in glomerular, blood vessel, or interstitial cells in vivo, whereas the rest of the serotypes showed no transduction. Conclusion: Since kidney-specific gene delivery via the renal artery by catheterization is highly feasible in humans, these findings provide useful information for promising strategies in renal gene therapy. Copyright © 2004 S. Karger AG, Basel.

BACKGROUND: To achieve human embryonic stem (ES) cell-based transplantation therapies, allogeneic transplantation models of nonhuman primates would be useful. We have prepared cynomolgus ES cells genetically marked with the green fluorescent protein (GFP). The cells were transplanted into the allogeneic fetus, taking advantage of the fact that the fetus is so immunologically immature as not to induce immune responses to transplanted cells and that fetal tissue compartments are rapidly expanding and thus providing space for the engraftment. METHODS: Cynomolgus ES cells were genetically modified to express the GFP gene using a simian immunodeficiency viral vector or electroporation. These cells were transplanted in utero with ultrasound guidance into the cynomolgus fetus in the abdominal cavity (n=2) or liver (n=2) at the end of the first trimester. Three fetuses were delivered 1 month after transplantation, and the other, 3 months after transplantation. Fetal tissues were examined for transplanted cell progeny by quantitative polymerase chain reaction and in situ polymerase chain reaction of the GFP sequence. RESULTS: A fluorescent tumor, obviously derived from transplanted ES cells, was found in the thoracic cavity at 3 months after transplantation in one fetus. However, transplanted cell progeny were also detected (approximately 1%) without teratomas in multiple fetal tissues. The cells were solitary and indistinguishable from surrounding host cells. CONCLUSIONS: Transplanted cynomolgus ES cells can be engrafted in allogeneic fetuses. The cells will, however, form a tumor if they "leak" into an improper space such as the thoracic cavity.

Transgenic (Tg) animals with reporter genes are useful models in which to study cell lineage and the process of differentiation into tissues. We developed the green fluorescent protein (GFP)-Tg rat, which is more suitable for transplantation and stem cell research because it is larger than mice. We found that marker gene expression was dependent on each organ and developmental stage. In this study we describe GFP expression in various tissues from embryonic, neonatal, and adult animals. GFP expression in brain, lung, liver, and islet tissues was restricted to early developmental stages, but it was continuously strong in the exocrine pancreas, kidney, and cardiac and skeletal muscles. The CAG promoter that was presumed to induce ubiquitous protein expression might be responsible for the differences in expression. (C) 2003 Wiley-Liss, Inc.

Dahl salt-sensitive rats are genetically hypersensitive to sodium intake. When fed a high sodium diet, they develop systemic hypertension, followed by cardiac hypertrophy and finally heart failure within a few months. Therefore, Dahl rats represent a good model with which to study how heart failure is developed in vivo. By using DNA microarray, we here monitored the transcriptome of >8000 genes in the left ventricular muscles of Dahl rats during the course of cardiovascular damage. Expression of the atrial natriuretic peptide gene was, for instance, induced in myocytes by sodium overload and further enhanced even at the heart failure stage. Interestingly, expression of the gene for the D-binding protein, an apoptotic-related transcriptional factor, became decreased upon the transition to heart failure. To our best knowledge, this is the first report to describe the transcriptome of cardiac myocytes during the disease progression of heart failure. (C) 2003 Elsevier Inc. All rights reserved.

Cell transplantation and regeneration therapy are potentially new therapeutic approaches for cardiovascular disease. Transgenic (tg) animals for reporter genes would be useful to follow the cell lineage and differentiation during development and regeneration processes. In the present study, we developed green fluorescent protein (GFP)-tg rats and evaluated them as a tool for the study of cardiomyocyte transplantation and regeneration. The myocardium and bone marrow cells derived from GFP-tg rats strongly expressed GFP. Because neonatal rat cultured cardiomyocytes also strongly expressed GFP, we transplanted GFP-tg rat-derived cardiomyocytes in a rat myocardial infarction (MI) model. Survival of GFP-tg rat-derived cardiomyocytes was confirmed. We further investigated whether bone marrow cells could differentiate into cardiomyocytes using this GFP-tg rat-derived bone marrow cells in vitro and in vivo. GFP-tg rat-derived bone marrow cells differentiated into cardiomyocyte- like cells (cardiac troponin I-expressed cells) by co-culture with wild rat cultured cardiomyocytes in vitro. Furthermore, differentiation of bone marrow cells into cardiomyocyte-like cells was observed by injection of GFP-tg rat-derived bone marrow cells in a rat MI model in vivo. These findings suggest that GFP-tg rats are a useful and valuable tool for the study of transplantation and regeneration in myocardium. © 2003 Plenum Publishing Corporation.

Animals transgenic (Tg) for reporter genes would be useful to following a given cell lineage during differentiation and regeneration processes. Here, we established a P-galactosidase (lacZ) Tg rat to use as a tool for regenerative research. Strong lacZ expression was observed in the skeletal muscles, myocardium, pancreas, and skin obtained from these lacZ-Tg rats, and moderate lacZ expression was observed in the liver, spleen, kidney, and cartilage. In contrast, brain, vessels, lung, adrenal gland, small intestine, blood leukocytes, bone marrow (BM) cells, and peripheral blood cells showed no lacZ expression. To test whether this lacZ-Tg rat could be used for regenerative research in myocardium, we induced myocardial injury after a lacZ-Tg BM transplant (BMT) into wild-type rats. The results show that lacZ-positive cardiomyocytes were found in the peri-infarct and uninjured myocardium in the BMT recipient rats. These findings suggest that lacZ-Tg rats are useful tool for regenerative research in the myocardium. (C) 2003 Elsevier Science (USA). All rights reserved.

Herpes simplex virus thymidine kinase (HSV-tk)/gancyclovir (GCV) therapy has the ability to inhibit tumor formation in animal models but the results of clinical trials have been disappointing. To improve the performance of tk/GCV therapy, we tried combination therapy designed to enhance its cytotoxic effects by introducing genes that induce apoptosis of the tumor cells through different pathways. We concentrated our efforts on the use of Bim, a BH3-only member of death activators in the Bcl-2 superfamily, because Bim is not involved in the pathways through which HSV-tk/GCV therapy induces apoptosis in malignant glioma cells. Among three alternative splicing variants, BimEL, BimL, and BimS, BimS lacks the binding domain for the dynein light chain LC8, which negatively regulates the proapoptotic function of BimEL and BimL. All four malignant glioma cell lines, U251, A172, T-430, and U373 underwent cell death after transfer of BimS using an adenovirus vector (AVC2). Intriguingly, combination of AVC2-BimS with AVC2-tk markedly increased the sensitivity of U251 cells to GCV both in vitro and in vivo. In contrast, AVC2-BimL did not induce significant cell death. These results indicated that BimS had the ability to improve the efficiency of HSV-tk/GCV therapy in the treatment of malignant glioma and suggested that the targeting of different proapoptotic pathways may be a useful strategy for the development of an effective gene therapy approach to treatment.

The application of adeno-associated virus (AAV) vectors to cancers is limited by their low transduction efficiency. Previously, we reported that gamma-ray enhanced the second-strand synthesis, leading to the improvement of the transgene expression, and cytocidal effect of the herpes simplex virus type-1 thymidine kinase (HSVtk) and ganciclovir (GCV) system. In this study, we extended this in vitro findings to in vivo. First, the laryngeal cancer cell line (HEp-2) and HeLa were treated with AAVtk/GCV, the number of surviving cells was reduced as the concentration of GCV increased. Furthermore, the 4 Gy irradiation enhanced the killing effects of AAVtk/GCV by four-fold on HeLa cells and 15-fold on HEp-2 cells. Following the in vitro experiments, we evaluated the transgene expression and the antitumor activity of the AA V vectors in combination with gamma-ray in nude mice inoculated with HEp-2 subcutaneously. The LacZ expression was observed in the xenografted tumors and significantly increased by gamma-ray. The AAVtk/GCV system suppressed the tumors growth, and gamma-ray augmented the antitumor activity by five-fold. These findings suggest that the combination of AAVtk/GCV system with radiotherapy is significantly effective in the treatment of cancers and may lead to reduction of the potential toxicity of both AAVtk/GCV and gamma-ray.

Perivascular macrophages are considered as cerebral scavenger cells under physical and pathological conditions. In this study, we tried to examine changes of perivascular macrophages, especially changes of the characteristic lysosomal inclusion bodies that are rich in hydrolytic enzymes, in the process of brain edema induced by cold injury. Wistar male rats aged 4 months were treated with dry ice for 20 minutes through a drilled hole at the parietal bone. At different time points after the cold injury, cerebral cortex was excised and the immunoreaction for cathepsin D, one kind of lysosomal protease, was examined by post-embedding immuno-electron microscopy. The reactions of cathepsin D were located in the inclusion bodies of perivascular macrophages. At 5 and 10 hours after cold injury, the reactions increased dramatically. Then the reactions inclined to decrease, and reached the minimum at 1 week after cold injury. The reactions seemed to recover at 2 weeks after cold injury. The changes of cathepsin D reactions suggest that the function of perivascular macrophages as scavenger cells were activated in the early phase of the process of brain edema, their later declines might be caused by severe pathological conditions.

Hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) have been shown to reduce atherosclerotic cardiovascular mortality and morbidity. Recent evidence indicates that statins may also exert direct effects on vascular wall cells (including endothelial cells and smooth muscle cells) independently of their hypocholesterolemic properties. However, little is known about whether statins have direct effects on myocardium. The effect of lipophilic and hydrophilic statins (fluvastatin and pravastatin) on apoptosis and protein synthesis in rat neonatal cardiac myocytes was investigated. The presence of apoptosis was evaluated by morphologic criteria, electrophoresis of DNA fragments, 4",6"-diamidine-2"-phenylindole (DAPI) staining, and TUNEL assay. Protein synthesis was measured by 3 H-leucine incorporation into the cells. Fluvastatin, but not pravastatin, induced apoptosis in cardiac myocytes in a time- and dose-dependent manner. The pro-apoptotic effect of fluvastatin was reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate (GGPP), but not in the presence of squalene. The addition of protein prenylation inhibitor perillic acid and Rho-kinase inhibitor Y27632 significantly increased apoptosis. Fluvastatin decreased RhoA protein in the membrane fraction, whereas there were no significant changes of the RhoA protein in the cytosol fraction. Interleukin-1beta-stimulated H-3-leucine incorporation was completely inhibited by fluvastatin, but not by pravastatin. The findings suggest that fluvastatin induces apoptosis in cardiac myocytes via protein prenylation and the subsequent inhibition of Rho, and may play a role in the pathogenesis of cardiac hypertrophy and remodeling.

We cloned a rat ABO homologue and established human A- and B-transferase transgenic rats. A DNA fragment corresponding to exon 7 of the human ABO gene was amplified from Wistar rat genomic DNA and sequenced. Using the amplified fragments as a probe for Southern blotting, multiple hybridized bands appeared on both EcoRI- and BamHI-digested genomes of seven rat strains, which showed variations in the band numbers among the strains. Four cDNAs were cloned from a Wistar rat, three of which showed A-iransferase activity and one of which showed B-transferase activity. These activities were dependent on the equivalent residues at 266 and 268 of human ABO transferase. Wild Wistar rats expressed A-antigen in salivary gland, intestine, and urinary bladder tissue, but B-antigen was not stained in any organs studied, whereas a transcript from the ABO homologue with B-transferase activity was ubiquitous. Human A-transferase and B-transferase were transferred into Wistar rats. A-transgenic rats expressed A-antigen in ectopic tissue of the brain plexus, type II lung epithelium, pancreas, and epidermis. B-antigen in the B-transgenic rat was expressed in the same organs as A-transgenic rats. These results may shed light on the function and evolution of the ABO gene in primates.

Vascular endothelial growth factor (VEGF), a bifunctional protein enhancing vascular permeability and stimulating endothelial growth, is thought to be responsible for fluid accumulation and angiogenesis in ascites tumors. To investigate the effects of stable expression of the soluble form of Fit-1 VEGF receptor (sFit-1), a known endogenous inhibitor of VEGF, on the malignant ascites tumors, we cotransduced RMG-1 human ovarian cancer cells with adeno-associated virus vectors carrying the sFlt-1 cDNA and Neo gene or Neo gene alone and isolated both the sFlt-1-expressing clone and the Neo-expressing clone. In vitro growth characteristics were essentially the same. As expected, conditioned medium collected from the sFlt-1-expressing cells significantly inhibited the human umbilical vein endothelial cell proliferation in the presence of recombinant VEGF. Expression of sFlt-1 significantly suppressed RMG-1 cell-induced angiogenesis in vivo in the mouse dorsal air sac assay model. We then inoculated sFlt-1- or Neo alone-expressing cells i.p. into female BALB/c nude mice. The average volume of ascites fluid, number of leaked RBCs, and number of cancer cells were significantly lower in mice injected with sFlt-1-expressing cells than in the controls. Survival time was significantly prolonged in mice injected with sFlt-1-expressing cells. These results suggest that inhibition of VEGF activity by sFlt-1 expression may provide a means to control carcinomatous ascites and angiogenesis of malignant ascites tumors.

Lysophosphatidylcholine (lysoPC) is a component of oxidized low density lipoprotein (LDL) and is involved in the pathogenesis of atherosclerosis and inflammation. Previous studies demonstrated that lysoPC can induce various protein kinases including tyrosine kinases, protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in vascular endothelial cells. However, the role of lysoPC-activated kinases remains undefined. In this study, we examined the effect of lysoPC on apoptosis and investigated the role of lysoPC-activated protein kinases in human umbilical vein endothelial cells (HUVEC). The presence of apoptosis was evaluated by morphological criteria, MTT assay, and electrophoresis of DNA fragments showing the characteristic apoptotic ladder, TUNEL analysis, and quantified as the proportion of hypodiploid cells by flow cytometry. The lysoPC induced apoptosis in a time- and dose-dependent manner. It stimulated the phosphorylation of extracellular signal-regulated kinasel/2 (ERK1/2) and p38-MAPK in HUVEC. The use of specific pharmacologic inhibitors indicated that the p38-MAPK-signaling pathway (SB203580) is required for lysoPC-induced apoptotic signals. Furthermore, lysoPC-induced apoptosis was inhibited by DEVD-FMK (a caspas-3/CPP32 inhibitor), suggesting involvement of an important segment in the apoptosis. These results demonstrate that lysoPC induces apoptosis in human endothelial cells through a p38-MAPK-dependent pathway. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) have been shown to attenuate proliferation of vascular smooth muscle cells (VSMCs) by mechanisms independent of lipid reduction. In the current study, we investigated the effect of lipophilic and hydrophilic statins (fluvastatin and pravastatin) on apoptosis in unstimulated or cytokine-stimulated VSMCs. The presence of apoptosis in rat VSMCs was evaluated by electrophoresis of DNA fragments and 4',6'-diamidine-2'-phenylindole staining and quantified by flow cytometry. Fluvastatin but not pravastatin enhanced apoptosis in interleukin-1beta-stimulated VSMCs. The proapoptotic effect of fluvastatin was fully reversed by mevalonate and geranylgeranyl-pyrophosphate, and partially by farnesyl-pyrophosphate, but not by squalene. Inhibition of the extracellular signal-regulated protein kinase (ERK1/2) pathway significantly increased fluvastatin-enhanced apoptosis, whereas inhibition of the p38-mitogen-activated protein kinase (MAPK) pathway significantly prevented this increase. However, fluvastatin showed no effect on the activity of ERK1/2 and p38-MAPK. Furthermore, fluvastatin-induced apoptosis was inhibited by YVAD-FMK (a caspase-1/interleukin-1beta-converting enzyme-like protease inhibitor) and DEVD-FMK (a caspase-3/CPP32 inhibitor), indicating involvement of an important segment in the apoptosis signaling pathway. These findings suggest that fluvastatin enhances apoptosis in cytokine-stimulated VSMCs and that protein prenylation, MAPK (ERK1/2 and p38-MAPK), and caspases are critically involved in the pathways of fluvastatin-enhanced apoptosis.

Mato, M., Takeuchi, K., Ookawara., S,Yamanaka, S., Mashiko, T., Ogura, K.:Inclusions in novel perivascular macrophages (Mato’s fluorescent granular perithelial cells) and neurons in the cerebral cortex of Hex A- and Hex B-deficient mice.Acta Neuropathologica 103:119-130, 2002

Beta-hexosaminidases are important enzymes for lipid and saccharide metabolism in the brain. In mice deficient in these enzymes, indigestible metabolic intermediates deposit in neurons. Inclusions such as membranous cytoplasmic bodies (MCB) and zebra bodies were seen in neurons of Tay-Sachs (TS) model mice, Sandhoff's disease (SD) model mice, and double knockout (DKO) mice. However, the cerebral perivascular macrophages discovered by Mato are active in the uptake of waste products and regarded as scavenger cells under steady-state conditions. We observed that indigestible components derived from neurons were taken up by the perivascular macrophages of TS mice by pinocytosis, but those of SD and DKO mice contained only pale inclusions and had marked vacuolations, and pinocytosis was rarely observed. Histochemically, the inclusions in the perivascular macrophages of TS mice were positive for the PAS stain, but those of SD and DKO mice were negative. In addition, the perivascular cells of TS mice expressed clear positive immunoreactivity against BM-8 and 74/80, but those of DKO mice had very weak BM-8 and F4/80 immunoreactivity. These differences between TS, SD, and DKO mice are based on their metabolism of oligosaccharides and glycosaminoglycans (GAG). Thus, hexosaminidase B is more important for keeping normal morphology and function of perivascular macrophages than hexosaminidase A. The foamy cells that appeared along the cerebral microvessels in lipidosis and saccharidosis were identified as perivascular macrophages (Mato's fluorescent granular perithelial cells: FGP cells).

Mato, M., Sakamoto, A., Ookawara, S., Takeuchi, K., Suzuki, K.: Ultrastructural and immunohistochemical changes of fluorescent granular perithelial cells and interaction of FGP cells and microglia after lipopolysaccharide administration. Anat. Rec. 251:330-338, 1998

Fluorescent granular perithelial (FGP) cells introduced by Mate et al. (1979, 1980, 1981) are situated in the Virchow-Robin space of cerebral microvessels and exhibit a marked uptake capacity for the endo- and exogenous materials in physiological conditions. That is, the FGP cells are the perivascular macrophage lineage enclosed by a basal lamina and glia limitans and are distributed between blood vessels and cerebral parenchyma. As reported previously, the FGP cells were distinguishable from microglia and pericytes in their morphology, location, and epitopes on their cytoplasmic membrane. On the other hand, it is established that microglia are upregulated by lipopolysaccaride (LPS) administration, but it remains unsettled whether the FGP cells, the perivascular macrophagic cells, are activated by LPS or not and what role the FGP cells play in the upregulation of microglia. Thus far, the morphological relationship between FGP cells and microglia in the process of upregulation has not been clarified. Thirty-six 7-month-old Wistar male rats were employed. The animals were sacrificed at 2, 5, 10, 24, and 72 h after the intravenous administration of 500 mu g/kg of LPS. The FGP cells and microglia in their cerebral cortex were studied with immunohistochemical and electron microscopical techniques. The findings of the present study indicated that the majority of FGP cells were upregulated by LPS from the results of immunohistochemical and morphological data, but some of them tended to degenerate. Furthermore, in the time course after LPS administration, microglia were also upregulated and approached the microvessels and occasionally contacted the FGP cells directly. From these findings, it is hypothesized that FGP cells may significantly influence the mobilization and upregulation of microglia caused by LPS administration. Anat. Rec. 251:330-338, 1998. (C) 1998 Wiley-Liss, Inc.

Dehydration increased the expression of aquaporin of collecting duct (AQP-2) and translocated AQP-2 to the apical plasma membranes from cytoplasmic vesicles of collecting duct cells. We determined whether the abrupt decrease in circulating arginine vasopressin (AVP) by giving excess water affects the expression of AQP-2 mRNA and subcellular localization of AQP-2 in collecting duct cells of the dehydrated rats. The 72-h water deprivation increased plasma AVP levels to 3.1 pg/ml and the expression of AQP-2 mRNA by 336% in rats, which were concomitantly abolished by the 40 ml/kg oral water load. A 50% inhibition of AQP-2 mRNA expression was obtained with 20 min after the forced water load. In immunohistochemistry and electron microscopy, the AQP-2 was manifestly present around the apical edge of collecting duct cells in the 72-h dehydrated rats. The AQP-8 was diffusely translocated into the cytoplasm 1 h after the forced water administration. These results indicate that AVP plays the on-off regulation of AQP-8 mRNA expression and that a majority of AQP-8 is regulated by the shuttle recycling in the collecting duct cells.

Asano, T., Ageyama, N., Takeuchi, K., Momoeda, M., Kitano, Y., Sasaki, K., Ueda, Y.,Suzuki, Y., Kondo, Y., Torii, R., Hasegawa, M., Ookawara, S., Harii, K., Terao, K., Ozawa, K., Hanazono, Y. : Engraftment and tumor formation after allogeneic in utero transplantation of primate embryonic stem cells. Transplantation. 76:1061-7, 2003

Takeuchi, K., Sereemaspun, A., Inagaki, T., Hakamata, Y., Kaneko, T., Murakami, T., Takahashi, M., Kobayashi, E., Ookawara, S.: Morphologic characterization of green fluorescent protein in embryonic, neonatal, and adult transgenic rats. Anat Rec. 274A:883-6, 2003

Ueno, S., Ohki, R., Hashimoto, T., Takizawa, T., Takeuchi, K., Yamashita, Y., Ota, J., Choi, YL., Wada, T., Koinuma, K., Yamamoto, K., Ikeda, U., Shimada, K., Mano, H. : DNA microarray analysis of in vivo progression mechanism of heart failure. Biochem Biophys Res Commun. 307:771-7, 2003

Kanazawa, T., Mizukami, H., Okada, T., Hanazono, Y., Kume, A., Nishino, H., Takeuchi, K., Kitamura, K., Ichimura, K., Ozawa, K.: Suicide Gene Therapy using AAV-HSVtk/ganciclovir in Combination with Irradiation Results in Regression of Human Head and Neck Cancer Xenografts in Nude Mice. Gene Ther. 10:51-8, 2003

Ogata, Y., Takahashi, M., Takeuchi, K., Ueno, S., Mano, H., Ookawara, S., Kobayashi, E., Ikeda, U., Shimada, K.: Fluvastatin induces apoptosis in rat neonatal cardiac myocytes: a possible mechanism of statin-attenuated cardiac hypertrophy. J Cardiovasc Pharmacol. 40:907-15, 2002