6, 7 In previous reports, our group was able to demonstrate a role for vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in the pathogenesis of portal hypertension, fibrosis, and HCC.8-10 Some studies suggest that angiogenesis plays a role in the progression of NASH. This was first brought to light in a study by Kitade et al.,11 which suggested that leptin-mediated neovascularization, coordinated by VEGF, is important in the development of liver fibrosis and HCC in a rat model for NASH. A macro-array gene expression analysis on the liver of

FK228 in vitro obese patients with severe NASH showed that VEGF, transforming growth factor beta, connective tissue growth factor, and fibroblast growth factor were overexpressed compared to control patients.12 Kitade et al.13 described a significant up-regulation of CD34 expression, which is widely used as a marker of neovascularization, in liver biopsies of patients with NASH. Recently, it was found that

VEGF is up-regulated in the serum of biopsy-proven steatosis and NASH patients compared to healthy controls.14, 15 These new findings could give a new perspective to investigate the pathophysiology of NASH. In this study we determined the role of angiogenesis at several timepoints in the pathophysiology of NASH in different mouse models. Moreover, we assessed whether inhibition of angiogenic factors could serve as a potential treatment of NASH. Therefore, we looked at the effect of anti-PlGF (αPlGF) and anti-VEGFR2 VX-765 in vivo (αVEGFR2) in vivo, using a prevention and treatment study in a mouse model for NASH,

and in vitro, using fat-laden primary hepatocytes. Ten-week-old C57BL/6 and homozygous db/db female mice (Charles River Laboratories, Brussels, Belgium) were kept under constant temperature and humidity on a 12-hour controlled dark/light cycle. Mice had ad libitum access to food and water. C57BL6/J and db/db mice were fed a methionine and choline-deficient (MCD) diet (MP Biomedicals, Brussels, Belgium) for 3 days, check details 1 week, 2 weeks, 4 weeks, or 8 weeks (n = 8/group) in order to develop NASH.16 Control groups received an identical diet to which choline bitartrate (2 g/kg) and DL-methionine (3 g/kg) was added (MP Biomedicals). Daily food intake was measured during the experiment. The Ethical Committee of Experimental Animals at the Faculty of Medicine and Health Sciences, Ghent University, approved the protocols. Anti-VEGFR2 (αVEGFR2; DC101) (ThromboGenics, Leuven, Belgium) and anti-PlGF (αPlGF; 5D11D4) (ThromboGenics) are known angiogenic inhibitors.17, 18 Anti-VEGFR2 (40 mg/kg body weight) and αPlGF (25 mg/kg body weight) were administered intraperitoneally to age- and weight-matched C57BL/6 mice for 8 weeks, two times a week (n = 10/group). Control mice were injected with phosphate-buffered saline (PBS) following the same dose and time schedule (n = 10/group).