1A-D). Inflammatory foci increased in the liver of C57BL5/J and db/db mice from 1 week of an MCD diet onwards compared to control mice. Furthermore, F4/80 staining significantly increased in C57BL6/J and db/db mice fed the MCD diet (P < 0.05) (Fig. 2A,B). Kupffer cells in animals fed a normal diet remained isolated, while the MCD diet caused recruitment of Kupffer cells into clusters (Supporting Fig. 2A,D). Increased tumor necrosis factor alpha (Tnf) and interleukin 1b (Il1b)
gene expression confirmed inflammation. We found a significant up-regulation of these inflammatory genes after 1 week in C57BL6/J ITF2357 in vivo and db/db mice till 8 weeks of the MCD diet compared to controls (P < 0.05) (Fig. 2C,D). Moreover, liver fatty acid binding protein 1 (L-fabp1), a gene involved in lipid transport, was significantly down-regulated in both mouse models after 4 weeks of MCD diet compared to controls (P < 0.05) (Fig. 2E,F). Stearoyl-CoA
desaturase 1 (Scd1), a gene involved in lipogenesis, was 500-fold less expressed in C57BL6/J mice and decreased 20-fold in db/db mice after 8 weeks of the MCD diet compared to controls (P < 0.001) (Fig. 2E,F). VEGF and PlGF are important angiogenic factors. VEGF expression was increased both in C57BL/6 and db/db fed an MCD diet. VEGF levels augmented after 3 days of MCD diet in db/db mice and after 1 week in C57BL6/J mice. In both mouse models the VEGF concentration selleck chemicals llc peaked after 4 weeks of the MCD diet (P < 0.001)
(Fig. 3A,B). Expression of CD105, an endothelial cell marker that is up-regulated by angiogenic factors such as VEGF, was significantly increased after 1 week of the MCD diet in C57BL6/J mice and after 3 days in db/db mice (P < 0.05) (Fig. 3C,D). In control mice, MCE CD105 showed a typical sinusoidal expression pattern. After 8 weeks of MCD diet, CD105 is more expressed in the liver tissue (Supporting Fig. 3A-D). Furthermore, gene expression of the Von Willebrand factor (Vwf) gene, another endothelial cell marker, increased steadily after 1 week in C57BL/6 mice on an MCD diet (Fig. 3E). Vwf gene expression in db/db mice peaks after 2 weeks of MCD diet (P < 0.001) (Fig. 3F). The vascular structure of C57BL6/J mice was determined by scanning electron microscopy images of vascular corrosion casts of the liver. These casts showed that mice fed an MCD diet have a more disrupted liver vasculature compared to controls (Fig. 6C,F). To address the role of angiogenic factors in the pathophysiology of NASH, a prevention (8 weeks αVEGFR2) and a treatment study (6 weeks αVEGFR2) were set up. In the prevention study, C57BL6/J mice were fed an MCD or control diet and were treated at the same time with VEGFR2 or PlGF antibodies for 8 weeks. In the treatment study, C57BL6/J mice started αVEGFR2 treatment after 2 weeks of MCD diet, when they had already developed steatosis, inflammation, and ballooning.