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Biomedical subjects

J Yoshii

Publications and source records attributed to J Yoshii.

At least 19 recordsLinked to original sources
PubMed2005

Angiopoietin 2 displays a vascular endothelial growth factor dependent synergistic effect in hepatocellular carcinoma development in mice.

BACKGROUND: Orchestration of two major classes of angiogenic factors-namely, vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2)-has been shown to play a pivotal role in tumour angiogenesis, including hepatocellular carcinoma (HCC). However, few studies have focused on the direct interaction of these factors on in vivo tumour development and angiogenesis. AIM: To examine the interaction between both factors in murine HCC. METHODS: We examined the combination effect of VEGF and Ang-2 overexpression by means of a combination of a retroviral tetracycline (tet) regulated gene manipulating system in vivo, by providing tet in the drinking water, and a conventional plasmid gene expression system. RESULTS: Neither Ang-2 nor VEGF overexpression induced proliferation of HCC cells in vitro. In vivo, although overexpression of Ang-2 did not increase tumour development, simultaneous expression of Ang-2 and VEGF synergistically augmented tumour growth and angiogenesis in murine HCC. Ang-2 plus VEGF induced tumour development was markedly attenuated by treatment with neutralising monoclonal antibodies against VEGF receptors. Ang-2 plus VEGF overexpression significantly increased the activities of matrix metalloproteinase (MMP)-2 and MMP-9 in the tumour. Suppression of intratumoral VEGF almost completely abolished this augmentation of MMPs. CONCLUSIONS: These results suggest that Ang-2 synergistically augments VEGF mediated HCC development and angiogenesis. This proangiogenic activity was exerted only in the presence of VEGF, at least partly mediated via induction of MMP-2 and MMP-9 in the tumour.

Angiopoietin-2↗
PubMed2003

Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis.

BACKGROUND: It has been shown that expression of the potent angiogenic factor, vascular endothelial growth factor (VEGF), and its receptors, flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), increased during the development of liver fibrosis. AIMS: To elucidate the in vivo role of interaction between VEGF and its receptors in liver fibrogenesis. METHODS: A model of CCl(4) induced hepatic fibrosis was used to assess the role of VEGFR-1 and VEGFR-2 by means of specific neutralising monoclonal antibodies (R-1mAb and R-2mAb, respectively). R-1mAb and R-2mAb were administered after two weeks of treatment with CCl(4), and indices of fibrosis were assessed at eight weeks. RESULTS: Hepatic VEGF mRNA expression significantly increased during the development of liver fibrosis. Both R-1mAb and R-2mAb treatments significantly attenuated the development of fibrosis associated with suppression of neovascularisation in the liver. Hepatic hydroxyproline and serum fibrosis markers were also suppressed. Furthermore, the number of alpha-smooth muscle actin positive cells and alpha1(I)-procollagen mRNA expression were significantly suppressed by R-1mAb and R-2mAb treatment. The inhibitory effect of R-2mAb was more potent than that of R-1mAb, and combination treatment with both mAbs almost completely attenuated fibrosis development. Our in vitro study showed that VEGF treatment significantly stimulated proliferation of both activated hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). VEGF also significantly increased alpha1(I)-procollagen mRNA expression in activated HSC. CONCLUSIONS: These results suggest that the interaction of VEGF and its receptor, which reflected the combined effects of both on HSC and SEC, was a prerequisite for liver fibrosis development.

Animals↗
PubMed2001

The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells.

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti-angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)-chelating agent for patients with Wilson's disease of penicillamine intolerance. In our study, we examined the effect of Cu-chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu-deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu-chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.

Animals↗
PubMed2001

The vascular endothelial growth factor receptor KDR/Flk-1 is a major regulator of malignant ascites formation in the mouse hepatocellular carcinoma model.

The vascular endothelial growth factor-A (VEGF-A), also known as the vascular permeability factor (VPF), has been shown to play an important role in malignant ascites formation. The effects of VEGF-A are mediated through flt-1 and kinase insert domain-containing receptor/fetal liver kinase (KDR/Flk-1) receptors. It has been shown that KDR/Flk-1 is a predominant receptor in solid hepatocellular carcinoma (HCC) development, but the role of this receptor in hepatic ascites formation has not yet been elucidated. In this study, we examined the role of KDR/Flk-1 in the murine MH134 hepatic malignant ascites formation by means of VEGF-A- and KDR/Flk-1-specific neutralizing antibodies (VEGF-A nAb and KDR/Flk-1 nAb, respectively). The mean volume of ascites, number of tumor cells in ascites, and the peritoneal capillary permeability were significantly suppressed by VEGF-A nAb and KDR/Flk-1 nAb treatment. These inhibitory effects of KDR/Flk-1 nAb were more potent than those of VEGF-A nAb. The autophosphorylation of KDR/ Flk-1 in the peritoneal wall was almost completely abolished by KDR/ Flk-1 nAb, whereas a certain level of activation was still shown by VEGF-A nAb treatment. Another VEGF-family, VEGF-C, which also binds KDR/Flk-1, was detected in the ascites. Furthermore, in the therapeutic experiment, although both VEGF-A nAb and KDR/Flk-1 nAb prolonged the survival rate of ascites-bearing mice, the latter showed a more significant impact on the survival of animals. These results suggest that KDR/Flk-1 is a major regulator of malignant hepatic ascites formation, and that in addition to VEGF-A, VEGF-C may also be involved in the malignant ascites formation via KDR/ Flk-1 activation.

Animals↗
PubMed2001

Angiotensin-II type 1 receptor interaction is a major regulator for liver fibrosis development in rats.

The renin-angiotensin system (RAS) is frequently activated in patients with chronic liver diseases. Angiotensin-II (AT-II) has been suggested to play an important role in liver fibrogenesis. It induces hepatic stellate cell (HSC) proliferation and up-regulates the transforming growth factor beta(1) (TGF-beta(1)) expression via AT-II type 1 receptor (AT(1)-R) in vitro. The aim of the present study was to examine the in vivo effect of candesartan (CA), a clinically used AT(1)-R blocker (ARB), and perindopril (PE), an angiotensin-converting enzyme (ACE) inhibitor (ACE-I), on pig serum-induced liver fibrosis development in rats. The clinically available comparable doses of CA and PE significantly attenuated the fibrosis development. These inhibitory effects of PE and CA were also found in the on-going liver fibrosis model. The hepatic hydroxyproline and serum fibrosis markers were significantly suppressed by CA and PE treatment. Furthermore, the alpha smooth muscle actin (alpha-SMA) positive cells in number were markedly suppressed by CA and PE treatment. Similarly, the hepatic TGF-beta(1) protein and messenger RNA (mRNA) levels were significantly suppressed. Our in vitro study showed that AT-II increased the TGF-beta(1) mRNA expression in the activated HSCs, and this effect was totally blocked by CA. These results suggested that the RAS, especially AT-II and AT(1)-R interaction plays a pivotal role in liver fibrosis development through HSC activation. Because both CA and PE are widely used in clinical practice without serious side effects, these drugs may provide an effective new strategy for anti-liver fibrosis therapy.

Angiotensin II↗
PubMed2001

The angiotensin-I-converting enzyme inhibitor perindopril suppresses tumor growth and angiogenesis: possible role of the vascular endothelial growth factor.

Angiotensin-I converting enzyme (ACE) inhibitor is used widely as an antihypertensive agent, and it has been suggested recently that it decreases the risk of cancer (A. F. Lever et al., Lancet, 352: 179-184, 1998). In this study, we examined the effect of several ACE inhibitors and angiotensin-II type 1 receptor (AT(1)-R) antagonists on tumor development and angiogenesis in a murine hepatocellular carcinoma model. Among ACE inhibitors, perindopril appeared to be a potent inhibitor of tumor development and angiogenesis, whereas AT(1)-R antagonists did not exert such an inhibitory effect. The inhibitory effect of perindopril was achieved even on established tumors. The level of the potent angiogenic factor, vascular endothelial growth factor (VEGF), in the tumor was significantly suppressed by perindopril. In vitro studies showed that perindopril-derived active form, perindoprilat, suppressed the endothelial cell tubule formation. Perindoprilat treatment also significantly inhibited VEGF mRNA expression in BNL-HCC cells in vitro. These results showed that the ACE inhibitor perindopril inhibited tumor development and angiogenesis independent from AT(1)-R blockage, and that VEGF alternation may be involved in the mechanism of this inhibitory effect. Because perindopril is widely used in clinical practice, it may represent an effective new strategy for anticancer therapy.

Angiotensin Receptor Antagonists↗
PubMed2000

Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to be increased in liver fibrosis development both in murine experimental models and human samples. However, the direct role of TIMP-1 during liver fibrosis development has not been defined. To address this issue, we developed transgenic mice overexpressing human TIMP-1 (hTIMP-1) in the liver under control of the albumin promoter/ enhancer. A model of CCl(4)-induced hepatic fibrosis was used to assess the extent of fibrosis development in TIMP-1 transgenic (TIMP-Tg) mice and control hybrid (Cont) mice. Without any treatment, overexpression of TIMP-1 itself did not induce liver fibrosis. There were no significant differences of pro-(alpha1)-collagen-I, (alpha2)-collagen-IV, and alpha-smooth muscle actin (alpha-SMA) mRNA expression in the liver between TIMP-Tg and Cont-mice, suggesting that overexpression of TIMP-1 itself did not cause hepatic stellate cell (HSC) activation. After 4-week treatment with CCl(4), however, densitometric analysis revealed that TIMP-Tg-mice had a seven-fold increase in liver fibrosis compared with the Cont-mice. The hepatic hydroxyproline content and serum hyaluronic acid were also significantly increased in TIMP-Tg-mice, whereas CCl(4)-induced liver dysfunction was not altered. An active form of matrix metalloproteinases-2 (MMP-2) level in the liver of TIMP-Tg-mice was decreased relative to that in Cont-mice because of the transgenic TIMP-1. Immunohistochemical analysis revealed that collagen-I and collagen-IV accumulation was markedly increased in the liver of CCl(4)-treated TIMP-Tg-mice with a pattern similar to that of alpha-SMA positive cells. These results suggest that TIMP-1 does not by itself result in liver fibrosis, but strongly promotes liver fibrosis development.

Actins↗
PubMed2000

BodyMap: a collection of 3' ESTs for analysis of human gene expression information.

BodyMap is a collection of site-directed 3' expressed sequence tags (ESTs) (gene signatures, GSs) that contains the transcript compositions of various human tissues and was the first systematic effort to acquire gene expression data. For the construction of BodyMap, cDNA libraries were made, preserving abundance information and histologic resolutions of tissue mRNAs. By sequencing 164,000 randomly selected clones, 88,587 GSs that represent chromosomally coded transcripts have been collected from 51 human organs and tissues. They were clustered into 18,722 independent 3' termini from transcripts, and more than 3000 of these were not found among ESTs assembled in UniGene (Build 75). Assessment of the prevalence of polyadenylation signals and comparison with GenBank cDNAs indicated that there was no significant contamination by internally primed cDNAs or genomic fragments but that there was a relatively high incidence (12%) of alternative polyadenylation sites. We evaluated the sensitivity and resolution of expression information in BodyMap by in silico Northern hybridization and selection of tissue-specific gene probes. BodyMap is a unique resource for estimation of the absolute abundance of transcripts and selection of gene probes for efficient hybridization-based gene expression profiling.

3' Untranslated Regions↗
PubMed1999

Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis.

The growth of any solid tumor depends on angiogenesis. Among the known angiogenic factors, vascular endothelial growth factor (VEGF) has been shown to play a pivotal role in tumor angiogenesis. However, to date, the signal transduction pathway initiated by VEGF is still not fully understood. It has been suggested that protein kinase C (PKC) plays an important role in the VEGF-induced signal transduction pathway in vitro, although the role of PKC in tumor angiogenesis in vivo still remains to be elucidated. By delivering the VEGF gene within the self-contained tetracycline-regulated retroviral vector (Retro-Tet) into hepatocellular carcinoma (HCC) cells, we manipulated VEGF expression by providing tetracycline in the drinking water to assess the tumor kinetics mediated exclusively by VEGF. In this study, we combined this Retro-tet system and LY333531, an inhibitor of the PKC-beta isoform, to elucidate the role of PKC-beta in tumor development and angiogenesis. Using a syngenic xenograft model, tumor augmentation induced by VEGF overexpression in HCC was markedly suppressed by oral administration of the PKC-beta inhibitor, with an accompanying reduction of neovascularization and p44/42 mitogen-activated protein kinase activation. This inhibitory effect was achieved even after the tumor was fully established. Immunohistochemical analysis revealed that apoptosis increased markedly in the tumor upon PKC-beta inhibitor treatment, whereas tumor cell proliferation itself did not change. Furthermore, with orthotopical transplantation, PKC-beta inhibition suppressed HCC tumor development in the liver. These results suggest that PKC-beta lies on the signal transduction pathway by which VEGF augments development and angiogenesis not only at the initial stage but also after the tumor is fully established.

Animals↗
PubMed1999

KDR/Flk-1 is a major regulator of vascular endothelial growth factor-induced tumor development and angiogenesis in murine hepatocellular carcinoma cells.

Vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors, has been shown to play a pivotal role in tumor angiogenesis, including hepatocellular carcinoma (HCC). The effects of VEGF are mediated mainly through two distinct receptors, flt-1 and KDR/Flk-1. It has been suggested that KDR/Flk-1 plays an important role in tumor development. However, the role of KDR/Flk-1 in HCC has not been examined. We previously reported that VEGF tightly regulated murine HCC development, based on the results of a study using a retroviral tetracycline-regulated (Retro-Tet) gene expression system. This system allows VEGF gene expression to be manipulated in vivo by providing tetracycline in the drinking water. In the present study, we combined the KDR/Flk-1-specific neutralizing monoclonal antibody (KDR/Flk-1mAb) and the Retro-Tet system to elucidate the role of KDR/Flk-1 in VEGF-induced tumor development and angiogenesis in a murine HCC experimental model. In a xenograft study, tumor augmentation induced by VEGF overexpression was almost abolished by means of KDR/Flk-1mAb treatment, with accompanying inhibition of angiogenesis, KDR/Flk-1 autophosphorylation, but not interference of flt-1 activation. This inhibitory effect was achieved even on established tumors and regardless of whether the tumor size was small or large. On the contrary, KDR/Flk-1mAb treatment significantly increased the apoptosis in the tumor. With orthotopic transplantation, KDR/Flk-1mAb also inhibited HCC development in the liver. These results suggest that KDR/Flk-1 is a major regulator of VEGF-mediated HCC development and angiogenesis not only at the initial stage, but also after the tumor has fully developed.

Animals↗
PubMed1998

Expression profile of active genes in granulocytes.

A number of genes active in granulocytes have been intensively studied as to the function of their products and their expression controls. However, the intensities and relative order of these gene activities have not been studied. This report describes an expression profile of 748 different species of active genes in human peripheral granulocytes obtained by analyzing a 3'-directed cDNA library that faithfully represents the mRNA population in the source cells. A significant fraction (20.3% of the total) of the expressed genes in granulocytes consisted of nuclear proteins such as DNA binding proteins, of secretory proteins such as cytokines, and of membrane proteins such as major histocompatibility complex (MHC) proteins and receptors. By comparing this expression profile with 11 profiles similarly obtained with unrelated human cells/tissues, we discovered 10 novel genes that are likely to act specifically in granulocytes. Comparison of this expression profile with that obtained with granulocytoids widely used as a granulocyte model by inducing a cultured promyelocytic leukemia cell line HL60 showed similarities and dissimilarities of gene expressions.

Adult↗
PubMed1998

Vascular endothelial growth factor tightly regulates in vivo development of murine hepatocellular carcinoma cells.

Angiogenesis is essential for the development of a solid tumor, including hepatocellular carcinoma (HCC). HCC is a well-known hypervascular tumor. Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors. Its role has not been clarified in vivo in HCC development. We used a self-contained, tetracycline-regulated retroviral vector system to elucidate the effect of VEGF on murine HCC development in a xenograft experimental model. By delivering the VEGF gene within the retroviral vector and under the control of a tetracycline-regulated promoter, we were able to manipulate VEGF expression in vivo tumor by providing tetracycline in the drinking water. Overexpression of VEGF showed a marked increase in tumor development accompanied by augmentation of neovascularization. The degree of tumor enlargement corresponded to the level of VEGF gene expression. Suppression of VEGF led to a decrease in tumor growth at the established tumor size, whether relatively small or large. The level of VEGF expression did not alter the proliferation of HCC cells in vitro. In a double-chamber chemoinvasion assay, the in vitro invasion activity of VEGF-transduced cells was not changed. In the presence of endothelial cells (EC), however, VEGF-transduced cells showed a marked increase in their in vitro invasion activity. These results suggested that VEGF plays a critical role in the development of HCC in cooperation with EC

Animals↗
PubMed1996

Identification and cloning of neuroblastoma-specific and nerve tissue-specific genes through compiled expression profiles.

An expression profile of active genes in a human neuroblastoma cell line CHP134 was obtained by collecting 1222 partial sequences from a 3'-directed cDNA library representing a non-biased mRNA population. By comparing this expression profile with the compiled profiles of multiple tissues, several novel gene transcripts that appeared only in the profile of the neuroblastoma cell line were identified. Further analyses by Northern blotting revealed two specific cDNA clones that are expressed in most of the human neuroblastomas examined, and three that are in some of the human neuroblastoma cell lines as well as in the adult human brain. Full-size cDNAs were cloned using these five partial cDNA sequences as probes and sequenced. A database search revealed that they are all novel and unique sequences: one sharing some amino acid sequence similarities with a cytoskeletal protein, two clones likely to be transcriptional factors, a clone that has characteristic potassium channel properties, and a clone that is non-homologous to any one of the known proteins. Thus, we argue that the collection of 3'-directed cDNA sequences in combination with the compiled expression profiles of active genes in multiple tissues is a powerful tool for discovering novel genes that are specifically expressed in a given cell or tissue, in this case neuroblastomas and/or nerve tissue.

Amino Acid Sequence↗
PubMed1995

Monitoring cell physiology by expression profiles and discovering cell type-specific genes by compiled expression profiles.

A gene expression profile is the list showing the expressed gene species and the abundance of their transcripts in a given cell or tissue. This list is made by constructing 3'-directed cDNA libraries consisting of only the 3'-termini of mRNA and sequencing randomly selected clones from such libraries: genes are identified by the sequences, and the composition of mRNA, which reflects gene activities, is measured from the frequency of appearance of the gene transcripts. For practical reasons, the number of sequenced clones has been limited to approximately 1000 per library at present, but the resulting profile covers almost all highly or moderately expressed genes, along with many less active genes. We constructed expression profiles from the HL60 human promyelocytic cell line and two of its derivatives, granulocytoids induced by DMSO and monocytoids induced by TPA. In HL60, a significant fraction of the abundantly expressed genes was for protein synthesis. Upon induction, these genes were partially or totally silenced; transcripts for proteins that characterize the granulocytes and monocyte-macrophages became abundant. By compiling and comparing different expression profiles, genes can be categorized into those expressed in diverse cell types and those active only in limited cell types. Although at present, the number of expression profiles that can be compiled is limited and this categorization is applicable only to abundantly expressed genes, 13 novel genes that may represent granulocyte- or monocyte-specific functions have been discovered.

Base Sequence↗
PubMed1994

Chromosomal assignments of 3'-directed partial cDNA sequences representing novel genes expressed in granulocytoid cells.

Large scale cDNA sequencing of 3'-directed cDNA libraries allows the collection of hundreds of novel sequences (gene signatures) and understanding of their expression levels in various tissues. Granulocytoid cells were induced from the human promyelocytic leukemia cell line 60 (HL60) with dimethyl sulfoxide (DMSO), and more than 1000 gene signatures were collected from a 3'-directed cDNA library of the granulocytoid cells. By selecting appropriate sequences for primer design, we systematically assigned chromosomes of these novel gene signatures by means of the polymerase chain reaction. We used a monochromosomal hybrid cell panel DNA as the template and localized 155 gene signatures to individual chromosomes. The results showed that 125 of them were assigned to single chromosomes and 30 to more than one. Genes assigned to more than one chromosome tend to be expressed ubiquitously, and their sequences are often conserved among humans and rodents. A highly sensitive laser fluorescent analyzer and ethidium staining were used for simple and large-scale PCR mapping.

Animals↗
PubMed1994

Chromosomal assignment of HepG2 3'-directed partial cDNA sequences by Southern blot hybridization using monochromosomal hybrid cell panels.

Large-scale sequencing of a 3'-directed cDNA library from the human liver cell line HepG2 has generated several hundred species of cDNA gene signatures, about 85% of which identify novel genes. They are useful molecular landmarks for human genome mapping. We used 160 of these novel signatures as probes for Southern hybridization to human DNA. We then identified the copy number of the corresponding genes and assigned them to chromosomes, with reference to monochromosomal hybrid cell mapping panels. The distribution profile of the expressing genes among chromosomes suggested that the expressing gene density is not uniform.

Animals↗
PubMed1994

An expression profile of active genes in human colonic mucosa.

An expression profile of genes active in the human colonic mucosa was obtained by collecting 959 partial sequences from a 3'-directed cDNA library. Seven genes were found to produce mRNA each of which comprised more than 1% of total mRNA. Four of these genes are novel, and are likely to be uniquely expressed in the colonic mucosa, and the other three have been identified as genes for fatty acid binding protein, immunoglobulin lambda chain, and carcinoma-associated antigen GA733-2. In the remaining 952 clones, 310 were composed of 118 species occurred recurrently but less than 1%, and 533 clones appeared only once. Because the 3'-directed cDNA library faithfully represents the mRNA population in the source tissue, these numbers represent the relative activities of the gene expression. Altogether 156 gene species were identified in GenBank, and a significant portion of these genes encode proteins found in Golgi apparatus and lysosomes, chromosome-encoded mitochondrial proteins, cell surface proteins, and components in the protein synthesis machinery. The types and proportions of genes identified is consistent with the known major activities of the colonic mucosa such as mucous protein production, energy-dependent water absorption, and rapid cell proliferation and turnover.

Aged↗