Volume 42, Issue 5, Pages 687-693 (May 2005) Combination of vitamin K2 and the angiotensin-converting enzyme inhibitor, perindopril, attenuates the liver enzyme-altered preneoplastic lesions in rats via angiogenesis suppression  Hitoshi Yoshiji, Shigeki Kuriyama, Ryuichi Noguchi, Junichi Yoshii, Yasuhide Ikenaka, Koji Yanase, Tadashi Namisaki, Mitsuteru Kitade, Masaharu Yamazaki, Tsutomu Masaki, Hiroshi Fukui  Journal of Hepatology  Volume 42, Issue 5, Pages 687-693 (May 2005) DOI: 10.1016/j.jhep.2004.12.025 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Effects of VK and PE on the development of DEN-induced GST-P and GGT-positive preneoplastic foci. The number (A) and size (B) of the GST-P positive foci were both significantly suppressed by the treatment of VK and PE. The inhibitory effect of PE was more potent than that of VK. The combination treatment of VK and PE exerted a much potent inhibitory effect as compared with that of PE. No positive foci were found in the PBS-treated group. As with the GGT-positive preneoplastic foci, the results were similar to those of the GST-P (Fig. 1C and D, respectively). No positive foci were found in the PBS-treated group. The results of the GGT-positive lesions were similar to those of the GST-P both in number (C) and size (D). The data represent means±SD. * and **, Statistically significant differences between the indicated groups (P<0.05 and 0.01), respectively. Journal of Hepatology 2005 42, 687-693DOI: (10.1016/j.jhep.2004.12.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 The effects of VK and PE on neovascularization in the liver. The CD31 mRNA expression was examined by real-time PCR as described in Section 2. The CD31 gene expression was significantly increased during hepatocarcinogenesis. Treatment with VK and PE significantly attenuated neovascularization in the liver. Suppression of angiogenesis by treatment with VK and PE was of similar magnitude to that of inhibition of development of the preneoplastic lesions. DEN, control DEN-treated rats (G1). PE and VK, PE- and VK-treated rats (G2 and G3, respectively). PE+VK, PE and VK combination-treated group (G4). PBS, PBS-injected negative control group (G5). The data represent the means±SD (n=15). * and **, statistically significant differences between the indicated groups (P<0.05 and 0.01, respectively). Journal of Hepatology 2005 42, 687-693DOI: (10.1016/j.jhep.2004.12.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Effects of VK on the HCC (A) and EC (B) cell proliferation in vitro. The cell proliferation was measured by MTT assay after harvest at 24, 48, 72, and 96h as described in Section 2. VK suppressed the in vitro proliferation of both HCC cell and EC in a dose-dependent manner. VK exerted a significant inhibitory effect on EC even at a dose of 1×10−6M, whereas this low dose did have any effect on HCC proliferation. At a dose of 1×10−4M, VK finally suppressed the in vitro proliferation of the HCC cells. PE had no effect on the HCC cells or EC even at a dose of 1×10−4M. Moreover, PE did not show any additional effect to that of VK on HCC cells and EC. The data represent the means±SD (n=6). *, Statistically significant differences as compared with the untreated group (P<0.01). HCC and EC, HepG2 and HUVEC, respectively. Journal of Hepatology 2005 42, 687-693DOI: (10.1016/j.jhep.2004.12.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Effects of VK on the in vitro EC tubular formation. (A) VK significantly inhibited the EC tubular formation. (B) Computer-assisted quantitative analysis showed that the inhibitory effect of VK on the EC tubular formation was in a dose dependent. The total tubule length was measured by an image analysis system as described in Section 2. The data represent the mean±SD (n=6). * and **, Statistically significant difference as compared to the untreated control group (P<0.05 and 0.01, respectively). Lane 1, untreated control group. Lanes 2–4, VK-treated group at the doses of 1×10−6, 1×10−5 and, 1×10−4M, respectively. Journal of Hepatology 2005 42, 687-693DOI: (10.1016/j.jhep.2004.12.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 The combination effect of VK and PE on the in vitro EC tubular formation. The tubules formed in the VK- or PE-treated group were less than in the untreated control group. The inhibitory effect of PE seemed to be stronger than that of VK (A). The quantitative analysis showed that the total length of the tubules formed in the VK- or PE-treated cultures was significantly less than in the untreated control culture, and that the combination treatment of PE and VK resulted in a further inhibition of the EC tubular formation as compared to PE alone (B). The total tubule length was measured by an image analysis system as described in Section 2. The data represent the mean±SD (n=6). * and **, Statistically significant difference between the indicated groups (P<0.05 and 0.01, respectively). Lane 1, untreated control group. Lanes 2 and 3, PE- and VK-treated group at the doses of 1×10−6, respectively. Lane 4, PE and VK combination-treated group. Journal of Hepatology 2005 42, 687-693DOI: (10.1016/j.jhep.2004.12.025) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions