-
Toru Nakamura,
Takuji Torimura,
Masaharu Sakamoto,
Osamu Hashimoto,
Eitaro Taniguchi,
Kinya Inoue, Ryuichiro Sakata,
Ryukichi Kumashiro,
Toyoaki Murohara,
Takato Ueno,
Michio Sata
[show abstract]
[hide abstract]
ABSTRACT: We investigated whether endothelial progenitor cell (EPC) transplantation could reduce established liver fibrosis and promote hepatic regeneration by isolating rat EPCs from bone marrow cells.
Recipient rats were injected intraperitoneally with carbon tetrachloride (CCl(4)) twice weekly for 6 weeks before initial administration of EPCs. CCl(4) was then readministered twice weekly for 4 more weeks, and EPC transplantation was carried out for these same 4 weeks.
At 7 days in culture, the cells expressed Thy-1, CD31, CD133, Flt-1, Flk-1, and Tie-2, suggesting an immature endothelial lineage. Immunohistochemical analyses showed fluorescent-labeled, transplantation EPCs were incorporated into the portal tracts and fibrous septa. Single and multiple EPC transplantation rats had reduced liver fibrosis, with decreased alpha2-(I)-procollagen, fibronectin, transforming growth factor-beta, and alpha-smooth muscle actin-positive cells. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area, in accordance with EPC location. Real-time polymerase chain reaction analysis of multiple EPC-transplantation livers showed significantly increased messenger RNA levels of matrix metalloproteinase (MMP)-2, -9 and -13, whereas tissue inhibitor of metalloproteinase-1 expression was significantly reduced. Expression of hepatocyte growth factor, transforming growth factor-alpha, epidermal growth factor, and vascular endothelial growth factor was increased in multiple EPC-transplantation livers, while hepatocyte proliferation increased. Transaminase, total bilirubin, total protein, and albumin levels were maintained in EPC-transplantation rats, significantly improving survival rates.
We conclude that single or repeated EPC transplantation halts established liver fibrosis in rats by suppressing activated hepatic stellate cells, increasing matrix metalloproteinase activity, and regulating hepatocyte proliferation.
Gastroenterology 08/2007; 133(1):91-107.e1. · 11.68 Impact Factor
-
Takuji Torimura,
Takato Ueno,
Motoaki Kin,
Eitaro Taniguchi,
Toru Nakamura,
Kinya Inoue, Ryuichiro Sakata,
Osamu Hashimoto,
Masaharu Sakamoto,
Hiromasa Ohira,
Ryukichi Kumashiro,
Michio Sata,
Hirohisa Yano,
Masamichi Kojiro,
Niina Veitonmaki,
Yihai Cao
[show abstract]
[hide abstract]
ABSTRACT: Recent studies indicate that kringle 1-5 has a potent and specific antiangiogenic activity. Here, we investigated the antitumor effect of kringle 1-5 gene transfer on hepatocellular carcinoma in mice.
The inhibitory effect of kringle 1-5 protein on proliferation of bovine capillary endothelial cells was evaluated by a tetrazolium-based assay. To study tumor growth, intrahepatic metastasis, and survival, liposome/kringle 1-5 complementary DNA complexes were injected intravenously in nude mice preimplanted with 1 of 3 hepatoma cell lines into the liver. Production of kringle 1-5 was tested by immunohistochemistry and Western blotting. Intratumoral vessel density was quantified. Expression of vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2 in tumors was examined by Western blotting. Serum alanine aminotransferase and alpha-fetoprotein levels and body weights were measured.
Proliferation of bovine capillary endothelial cells was inhibited by purified kringle 1-5 in a dose-dependent manner. Gene transfer of kringle 1-5 caused a significant reduction in vessel density with suppression of tumor growth of the 3 hepatoma cell lines and serum alpha-fetoprotein levels, prolonged the survival period, and reduced the number of intrahepatic metastases. Among the analyzed angiogenic factors, kringle 1-5 reduced angiopoietin-2 expression levels. Expression of kringle 1-5 protein was detected on hepatoma cells and hepatocytes in the liver. However, it did not alter serum alanine aminotransferase levels and body weights, suggesting kringle 1-5 lacks severe side effects.
Antiangiogenic gene therapy with kringle 1-5 complementary DNA is a promising safe and effective strategy for suppression of growth of hepatocellular carcinoma.
Gastroenterology 05/2006; 130(4):1301-10. · 11.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The concentration of plasma vitronectin was determined and compared with various parameters of liver function including the blood coagulation system in patients with liver diseases. The severity of cirrhosis was graded according to Child's criteria and compared with the plasma vitronectin level. Furthermore, the distribution of vitronectin in the liver of patients with liver diseases was studied by light and electron microscopy using the indirect immunoperoxidase method.The plasma vitronectin level was low in all liver disease groups as compared with the healthy controls. The difference from the controls was significant in patients with hepatocellular carcinoma and decompensated cirrhosis. Moreover, the plasma vitronectin level was positively correlated with the levels of serum cholinesterase, albumin, plasma α2 plasmin inhibitor-plasmin complex and the prothrombin time and results of the hepatoplastin test. Plasma vitronectin decreased with increasing severity of cirrhosis according to Child's criteria. These results suggest that the plasma vitronectin level is a useful parameter of hepatic synthetic function in patients with liver diseases; it may also reflect the severity of cirrhosis.Light microscopy revealed vitronectin in the area of focal necrosis and the portal tracts in the liver of patients with acute viral hepatitis, in the area of piecemeal necrosis in the liver of patients with chronic hepatitis and along the area of fiber deposition in the liver of patients with cirrhosis. Immunoelectron microscopy showed vitronectin in the rough endoplasmic reticulum of hepatocytes. Moreover, vitronectin was seen around inflammatory cells, endothelial cells, Ito cells and hepatocytes in the perisinusoidal area near focal necrosis and piecemeal necrosis and on collagen fibers.These results suggest that vitronectin may be produced by hepatocytes and that they play an important role as an extracellular matrix component in the injured liver. (Hepatology 1992;15:629–636).
Hepatology 12/2005; 15(4):629 - 636. · 11.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To determine the effects of dominant-negative TGF-beta receptor expression during liver regeneration in rats with dimethylnitrosamine (DMN)-induced liver injury.
Rats were first treated with DMN for 3 weeks, and then intravenously injected once with AdTbeta-TR, AdLacZ, or saline. Serial changes in hepatocyte proliferation and apoptosis were evaluated by immunohistochemistry using anti-Ki67 antibody, and TUNEL staining, respectively. The mRNA expression of regeneration factors (HGF, TGF-alpha, EGF, and IGF-I) and IL-6 were evaluated by real-time PCR and northern blotting.
Anti-TGF-beta molecular intervention up-regulated hepatocyte proliferation and inhibited apoptosis. In the AdTbeta-TR-treated rats, EGF and IGF-I mRNA expression levels were significantly increased at day 1 and remained high for 3 days after gene transfer; TGF-alpha mRNA expression levels were significantly increased at 2 to 5 days after gene transfer; HGF mRNA expression levels were significantly up-regulated at day 2 only after gene transfer; while IL-6 mRNA expression level tended to increase at day 1, but decreased thereafter.
In rats with DMN-induced liver injury, anti-TGF-beta molecular intervention therapy stimulates proliferation and reduces apoptosis of hepatocytes, and also up-regulates the transcription of various growth factors.
Journal of Hepatology 01/2005; 41(6):974-82. · 9.26 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hepatic stellate cells (HSC) are central to liver fibrosis. The eicosanoid pathway and cyclooxygenase-2 (COX-2) may be an important signaling mechanism in HSC. We investigated the role of COX-2, prostaglandin E(2) (PGE(2)) and prostaglandin I(2) (PGI(2)) in proliferation of LI90, an immortalized cell line of HSC. Our results showed that COX-2 was upregulated by platelet-derived growth factor (PDGF), a mitogen in HSC. COX-2 was responsible for the production of PGE(2) and PGI(2) in PDGF-stimulated LI90 cells. Furthermore, we demonstrated that COX-2 and PGE(2) mediated the proliferative response of LI90 to PDGF while synthetic analogue of PGI(2) exhibited anti-proliferative effect. Our findings suggest complex interactions of prostaglandins in liver fibrogenesis. In vivo studies using animal models are needed to elucidate the effect of COX-2 inhibition by non-steroidal anti-inflammatory drugs or COX-2 inhibitor in hepatic fibrosis.
Prostaglandins Leukotrienes and Essential Fatty Acids 12/2004; 71(5):329-33. · 3.37 Impact Factor
-
Takuji Torimura,
Takato Ueno,
Motoaki Kin,
Riko Harada,
Eitaro Taniguchi,
Toru Nakamura, Ryuichiro Sakata,
Osamu Hashimoto,
Masaharu Sakamoto,
Ryukichi Kumashiro,
Michio Sata,
Osamu Nakashima,
Hirohisa Yano,
Masamichi Kojiro
[show abstract]
[hide abstract]
ABSTRACT: Hepatocellular carcinoma (HCC) is a highly vascular tumor. Angiopoietin-1 and Angiopoietin-2 have been shown to be involved in tumor angiogenesis. We investigated the expression of Angiopoietin-1 and Angiopoietin-2 in HCC.
The expression of Angiopoietin-1 and Angiopoietin-2 mRNAs in cultured hepatoma cells under hypoxic conditions and in HCC and noncancerous liver tissue was evaluated by real-time PCR. The expression of Angiopoietin-1, Angiopoietin-2, and their receptor Tie-2 in HCC was assessed by immunohistochemistry. The changes in Angiopoietin-1 and Angiopoietin-2 expression were evaluated in relation to tumor differentiation and changes in tumor vascularity.
Hypoxic conditions did not up-regulate the expression of Angiopoietin-1 and Angiopoietin-2 mRNAs in hepatoma cells. Increased expression of Angiopoietin-1 and Angiopoietin-2 mRNAs was detected in HCC. Angiopoietin-1 and Angiopoietin-2 were detected in hepatoma cells, hepatic stellate cells, and smooth muscle cells, whereas Tie-2 was detected in endothelial cells, hepatic stellate cells and smooth muscle cells. Increased expression of Angiopoietin-2 and Angiopoietin-2 mRNA was associated with tumor dedifferentiation. The expression of Angiopoietin-1 and Angiopoietin-2 correlated with HCC vascularity.
Our findings indicate that the increased expression of Angiopoietin-1 and Angiopoietin-2 play a critical role in the process of vascular development in HCC.
Journal of Hepatology 06/2004; 40(5):799-807. · 9.26 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Green-tea polyphenols are known to have anti-fibrotic properties of the skin and the artery. The proliferation of hepatic stellate cells (HSC) is closely related to the progression of liver fibrosis in chronic liver diseases. We investigated the inhibitory effect of epigallocatechin-3-gallate (EGCG), the major potential inhibitory component of green-tea polyphenols, on the proliferation of HSC. The aim of this study was to clarify the molecular mechanisms of EGCG inhibition of HSC proliferation.
A cultured human hepatic stellate cell line LI90 was used for this study. The cells were stimulated by platelet-derived growth factor (PDGF)-BB in the presence or absence of EGCG. Proliferation was determined by bromodeoxy-uridine incorporation. The mRNA expressions of collagen alpha1(I) and (IV) were evaluated by a quantitative reverse transcription-polymerase chain reaction. PDGF receptor tyrosine phosphorylation was detected using anti-phosphotyrosine antibody. PDGF receptor radioligand binding assay was performed by [125I]-PDGF-BB.
EGCG inhibited the PDGF-BB-induced cell-proliferation and collagen alpha1(I) and (IV) mRNA expressions. EGCG reduced the autophosphorylation of the PDGF receptor. EGCG blocked PDGF-BB binding to its receptor in a non-competitive manner.
EGCG has an inhibitory effect on PDGF-induced proliferation of HSC, and the blocking of PDGF-BB binding to its receptor may be the mechanism behind this effect.
Journal of Hepatology 02/2004; 40(1):52-9. · 9.26 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We investigated whether anti–transforming growth factor β (TGF-β) molecular intervention can halt the progression of liver fibrosis in rats. To block TGF-β action in a specific manner, we prepared an adenovirus expressing a truncated type II TGF-β receptor (AdTβ-TR), which specifically inhibits TGF-β signaling as a dominant-negative receptor. We also used an adenovirus expressing bacterial β-galactosidase (AdLacZ) as a control adenovirus. Rats were treated with dimethylnitrosamine (DMN) for 3 weeks; then, AdTβ-TR, AdLacZ, or saline was intravenously applied once, followed by an additional 3-week DMN treatment. The ratio between the truncated receptor and the wild-type receptor at the mRNA level was 15 at 1 week and 10 at 3 weeks after gene transfer. Immunohistostaining analysis showed that the truncated receptor was expressed mainly in septal cells including hepatic stellate cells. Liver fibrosis, as assessed by histology, hydroxyproline content, and the serum level of hyaluronic acid, progressed during the additional 3-week DMN treatment. However, in rats infected with AdTβ-TR, the fibrosis remained at the level seen in rats given DMN for only 3 weeks. All AdTβ-TR–treated rats remained alive, whereas DMN-treated rats infused with either AdLacZ or saline died of liver dysfunction. In the livers of AdTβ-TR–treated rats, electron microscopy showed: 1) less accumulation of extracellular matrix proteins in the Disse's spaces; 2) regenerated hepatocytes; and 3) fat droplet–rich “quiescent” hepatic stellate cells. Our results demonstrate that TGF-β plays a critical role in the progression of liver fibrosis, and suggest that anti–TGF-β intervention should be therapeutic in already-established fibrotic livers, not only by suppressing fibrosis, but by facilitating hepatocyte regeneration.
Hepatology 07/2000; 32(2):247 - 255. · 11.66 Impact Factor
