Junichi Yoshii

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 CCl4 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 CCl4, 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 α-smooth muscle actin positive cells and α1(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 α1(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.

Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats†

Nonalcoholic steatohepatitis (NASH) may cause fibrosis, cirrhosis, and hepatocellular carcinoma (HCC); however, the exact mechanism of disease progression is not fully understood. Angiogenesis has been shown to play an important role in the progression of chronic liver disease. The aim of this study was to elucidate the role of angiogenesis in the development of liver fibrosis and hepatocarcinogenesis in NASH. Zucker rats, which naturally develop leptin receptor mutations, and their lean littermate rats were fed a choline‐deficient, amino acid–defined diet. Both Zucker and littermate rats showed marked steatohepatitis and elevation of oxidative stress markers (e.g., thiobarbital acid reactive substances and 8‐hydroxydeoxyguanosine). In sharp contrast, liver fibrosis, glutathione‐S‐transferase placental form (GST‐P)‐positive preneoplastic lesions, and HCC developed in littermate rats but not in Zucker rats. Hepatic neovascularization and the expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, only increased in littermate rats, almost in parallel with fibrogenesis and carcinogenesis. The CD31‐immunopositive neovessels were mainly localized either along the fibrotic septa or in the GST‐P–positive lesions. Our in vitro study revealed that leptin exerted a proangiogenic activity in the presence of VEGF. In conclusion, these results suggest that leptin‐mediated neovascularization coordinated with VEGF plays an important role in the development of liver fibrosis and hepatocarcinogenesis in NASH. (HEPATOLOGY 2006;44:983–991.)

TISSUE INHIBITOR OF METALLOPROTEINASES-1 (TIMP-1) INHIBITS TUMOR GROWTH AND ANGIOGENESIS IN THE TIMP-1 TRANSGENIC MOUSE MODEL

The tissue inhibitor of matrix metalloproteinases‐1 (TIMP‐1) has been recognized as a multifunctional protein. The role of TIMPs in cancer remains the subject of conflicting reports with an antitumor activity or a tumor growth stimulation activity by several mechanisms. The aim of our study is to investigate the effect of ectopic TIMP‐1 overexpression on the primary transplanted tumor growth. We employed transgenic mice overexpressing the human TIMP‐1 (hTIMP‐1) in the liver under control of the albumin promoter/enhancer (TIMP‐Tg‐mice) and producing high serum levels of TIMP‐1. We used the transplantable Ehrlich tumor cells in the current study. The allograft study revealed that the tumor growth in the TIMP‐Tg‐mice was more significantly inhibited than control (Cont) mice by associated suppression of neovascularization in the tumor. The in vitro studies showed that the recombinant TIMP‐1 (rTIMP‐1) did not affect the proliferation of the endothelial cells (ECs) and tumor cells, suggesting that the tumor suppressive effect of TIMP‐1 was not due to cytotoxicity. TIMP‐1 significantly inhibited EC tubular formation in vitro. Furthermore, TIMP‐1 treatment did not affect the levels of matrix metalloproteinase (MMP)‐2 and MMP‐9 mRNA in the Ehrlich tumor cells in vitro, although these expressions in the tumor were markedly suppressed in the TIMP‐Tg‐mice, compared to the Cont‐mice at the end of the experiment. These results suggested that the ectopically overexpressed TIMP‐1 inhibited the tumor growth by angiogenesis suppression.

Halting the interaction between vascular endothelial growth factor and its receptors attenuates liver carcinogenesis in mice

It has been shown that angiogenesis plays an important role not only in tumor growth, but also in early carcinogenesis. The expression of a potent angiogenic factor, vascular endothelial growth factor (VEGF), increased during the early stage of carcinogenesis. In this study, the effects of the neutralizing monoclonal antibodies R1 mAb and R2 mAb of the VEGF receptors Flt‐1 (VEGFR‐1) and KDR/Flk‐1 (VEGFR‐2), respectively, on murine hepatocarcinogenesis induced by diethylnitrosamine (DEN) were examined. The effects of R1 mAb and R2 mAb on spontaneous lung metastasis from hepatocellular carcinoma (HCC) were also investigated. VEGF expression and neovascularization in the tumor increased stepwise during hepatocarcinogenesis. Treatment with both R1 mAb and R2 mAb markedly inhibited the development of HCC and adenoma in the liver. The inhibitory effect of R2 mAb was more potent than that of R1 mAb, and the combination treatment with both mAbs almost completely attenuated hepatocarcinogenesis. Both R1 mAb and R2 mAb treatment significantly suppressed the development of angiogenesis in HCC. The suppressive effects against angiogenesis R1 mAb and R2 mAb were similar in magnitude to their inhibitory effects against hepatocarcinogenesis. Furthermore, spontaneous lung metastasis from HCC was also significantly suppressed by R1 mAb and R2 mAb treatment. In conclusion, these results suggest that VEGF and receptor interaction plays an important role in hepatocarcinogenesis and in spontaneous lung metastasis from HCC. (HEPATOLOGY 2004;39:1517–1524.)

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 Wilsons 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.

Inhibition of renin–angiotensin system attenuates liver enzyme-altered preneoplastic lesions and fibrosis development in rats

BACKGROUND/AIMS It is suggested that the renin-angiotensin system (RAS) is involved in tumor development and fibrogenesis. The aim of the present study was to examine the effect of RAS inhibition on the liver enzyme-altered preneoplastic lesions and fibrosis development. METHODS The effects of the clinically used angiotensin-I converting enzyme inhibitor (ACE-I), perindopril (PE), on two different rat model of liver carcinogenesis models induced separately by diethylnitrosamine (DEN) and a choline-deficient L-amino acid-defined (CDAA) diet were studied. This CDAA model was also used to elucidate the effect of PE on liver fibrosis development. RESULTS The immunohistochemical evaluation revealed that the glutathione S-transferase placental form (GST-P), and gamma-glutamyltransferase (GGT)-positive preneoplastic foci significantly decreased in the livers of the PE-treated groups. In CDAA-induced liver fibrosis model, PE revealed a marked inhibitory effect of liver fibrosis development. The hepatic hydroxyproline, serum fibrosis markers, alpha-smooth muscle actin (alpha-SMA) immunopositive cells in number, and alpha-(III) pro-collagen mRNA expression were significantly suppressed by PE treatment. These inhibitory effects of PE were achieved even at a clinically comparable dose (2 mg/kg per day). CONCLUSIONS These results suggested that the RAS is involved in liver carcinogenesis and fibrosis development.

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.

Dipeptidyl peptidase-4 inhibitor attenuates hepatic fibrosis via suppression of activated hepatic stellate cell in rats

BackgroundDipeptidyl peptidase-4 inhibitor (DPP4-I) is clinically used as a new oral antidiabetic agent. Although DPP4 is reportedly associated with the progression of chronic liver diseases, the effect of DPP4-I on liver fibrosis development is still obscure. This study was designed to elucidate the effect of DPP4-I on liver fibrosis development in conjunction with the activated hepatic stellate cells (Ac-HSCs).MethodsThe antifibrotic effect of DPP4-I was assessed in vivo and in vitro using porcine serum-induced experimental liver fibrosis model. DPP4-I, sitagliptin, at a clinically comparable low dose was administered by gavage daily.ResultsDPP4-I significantly attenuated liver fibrosis development along with the suppression of hepatic transforming growth factor (TGF)-β1, total collagen, and tissue inhibitor of metalloproteinases-1 in a dose-dependent manner. These suppressive effects occurred almost concurrently with the attenuation of HSCs activation. Our in vitro studies showed that DPP4-I inhibited platelet-derived growth factor-BB-mediated proliferation of the Ac-HSCs as well as upregulation of TGF-β1 and α1(I)-procollagen at magnitudes similar to those of the in vivo studies. The inhibitory effects of DPP4-I against HSCs proliferation and fibrogenic gene expression are mediated through the inhibition of the phosphorylation of ERK1/2, p38 and Smad2/3, respectively.ConclusionsDPP4-I markedly inhibits liver fibrosis development in rats via suppression of HSCs proliferation and collagen synthesis. These suppressive effects are associated with dephosphorylation of ERK1/2, p38 and Smad2/3 in the HSCs. Since DPP4-I is widely used in clinical practice, this drug may represent a potential new therapeutic strategy against liver fibrosis in the near future.

Branched-chain amino acids suppress insulin-resistance-based hepatocarcinogenesis in obese diabetic rats

BackgroundBranched-chain amino acids (BCAAs) reportedly inhibit the incidence of hepatocellular carcinoma (HCC) in patients with liver cirrhosis and obesity that is frequently associated with insulin resistance (IR). However, the possible mechanism is still obscure. The aim of the present study was to examine the effect of BCAAs, especially in conjunction with angiogenesis, on hepatocarcinogenesis under the condition of IR.MethodsThe effect of BCAAs on the development of liver enzyme-altered preneoplastic lesions and angiogenesis was examined in obese diabetic Otsuka Long-Evans Tokushima Fatty rats. We also performed an in vitro study to elucidate the possible mechanisms involved.ResultsTreatment with BCAAs markedly inhibited glutathione-S-transferase placental form (GST-P)-positive preneoplastic lesions along with suppression of neovascularization in the liver. The hepatic expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, was also attenuated. BCAA treatment significantly suppressed glucose- and insulin-induced in vitro angiogenesis in the presence of VEGF.ConclusionsIn obese diabetic rats BCAAs exerted a chemopreventive effect against HCC, associated with the suppression of VEGF expression and hepatic neovascularization. Since BCAA preparations are widely used in clinical practice for patients with chronic liver diseases, this agent may represent a new strategy for chemoprevention against HCC in the future.

Losartan, an angiotensin-II type 1 receptor blocker, attenuates the liver fibrosis development of non-alcoholic steatohepatitis in the rat

BackgroundApart from simple steatosis, the non-alcoholic steatohepatitis (NASH) can progress into liver fibrosis and cirrhosis. To date, however, no widely accepted therapeutic modalities have been established against NASH in the clinical practice. To find out promising new therapeutic agents, it is important to employ an appropriate experimental model of NASH, such as association with insulin resistance.FindingsIn the current study, we found that losartan, a clinically used angiotensin-II type 1 receptor blocker, significantly attenuated a choline-deficient L-amino acid-defined (CDAA) diet-induced steatohepatitis in obese diabetic- and insulin resistance-associated Otsuka Long-Evans Tokushima Fatty (OLETF) rats. The transforming growth factor-beta, a well-known major fibrogenic cytokine, was also suppressed in a similar magnitude to that of the fibrosis area. Noteworthy was the finding that these inhibitory effects were achieved even at a clinically comparable low dose.ConclusionSince losartan is widely used without serious side effects in the clinical practice, this agent may be an effective new therapeutic strategy against NASH.