05). Serum levels of ALT, AST, triglyceride (TG), and cholesterol did not differ between untreated, 6 weeks αVEGFR2, 8 weeks αVEGFR2, and αPlGF
treated groups (Table 2). Steatosis, inflammation, ballooning, and fibrosis were assessed histologically using H&E and Sirius Red staining (Fig. 4A). As illustrated in representative sections, click here the liver of mice fed an MCD diet and treated with αVEGFR2 during 8 weeks had significantly lower grades of steatosis and inflammation compared to the PBS-treated group. Mice treated for 6 weeks with αVEGFR2, in a preventive setting, also showed significantly less steatosis and inflammation compared to untreated mice. This clearly shows that αVEGFR2 prevents the progression to NASH both in a preventive and a therapeutic setting. The liver of mice treated with αPlGF showed no significant changes in liver histology compared to the PBS-treated group (Fig. 4B-F). The presence of inflammatory infiltrates in the liver was examined with F4/80 staining. The staining showed that Kupffer cells were more isolated and formed fewer clusters in mice treated with 6 or 8 weeks of αVEGFR2 compared to MCD-fed mice treated with PBS (Supporting Fig. 2A,B,E-G). Mice treated with αVEGFR2 for 8 weeks had significantly less F4/80 staining compared to untreated
mice (Fig. Fulvestrant cell line 5A). Gene expression in the liver of Tnf and Il1b gene confirmed that αVEGR2 treatment reduced inflammation in the liver of MCD-fed mice for 8 weeks compared to mice treated with PBS (Fig. 5B). Scd1 gene expression was significantly increased in mice treated with αVEGFR2 for 6 and 8 weeks compared to mice treated with PBS (P < 0.001) (Fig. 5C). Expression of L-fabp1, a gene involved in lipid
transport, was not affected by any treatment (Fig. 5C). Lipid regulation in vitro was assessed by AdipoRed assay. Dose-response curves showed that a concentration of 100 μg αVEGFR2/mL was optimal and showed that MCE αVEGFR2 therapy significantly decreased lipid accumulation in fat-laden primary hepatocytes (Fig. 5D,E). Expression of CD105 was significantly decreased in C57BL/6 mice on 8 weeks of an MCD diet and treated with 6 or 8 weeks of αVEGFR2 compared to MCD-fed mice treated with PBS (Fig. 6A) (Supporting Fig. 3A,B,E-G). Only the group treated for 8 weeks with αVEGFR2 showed a reduced expression of the Vwf gene compared to untreated MCD fed mice (P < 0.001) (Fig. 6B). CD105 and Vwf gene expression of mice on 8 weeks of an MCD diet compared to mice on a control diet were increased in the αPlGF and PBS-treated group, confirming our previous data (Figs. 3C,E; 6A,B). Hepatic stellate cell (HSC) activation was evaluated with alpha-smooth muscle actin (αSma) gene expression in the liver and visualized with an αSMA staining. Gene expression of αSMA was significantly up-regulated in mice fed the MCD diet. Anti-VEGFR2 treatment for 6 or 8 weeks significantly reduced αSMA expression (Fig. 6B).