Increases in several fibrogenic genes were confirmed in VhlF/F;Al

Increases in several fibrogenic genes were confirmed in VhlF/F;AlbERcre mice treated with tamoxifen, compared to littermate control mice (Fig. 6A). A specific increase in lysyl oxidase-like 1 (LOXL1), lysyl oxidase-like Cell Cycle inhibitor 2 (LOXL2), prolyl 4-hydroxylase alpha 1 (P4HA1), prolyl 4-hydroxylase alpha 2 (P4HA2), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), and transglutaminase 2 (TGM2) was observed. These genes are critical for the formation and stabilization

of collagen.21-26 In addition, smooth muscle actin (SMA), a marker of stellate cell activation and fibrosis, was significantly increased in VhlF/F;AlbERcre mice treated with tamoxifen, compared to littermate control mice, as assessed by qRT-PCR and western blot analysis (Fig. 6A,B). To confirm an increase in fibrosis, Masson’s trichrome staining was performed (Fig. 6C,D). Livers isolated from VhlF/F;AlbERcre mice 14 days after tamoxifen treatment demonstrated a moderate increase in focal areas of fibrosis, compared to similarly treated VhlF/F mice (Fig. 6C). Moreover, VhlF/F and VhlF/F;AlbERcre mice were treated with tamoxifen, then put on liquid diet consisting of 4% ethanol for 2 weeks. Mice are resistant to alcohol-induced fibrosis, as chronic treatment with alcohol (i.e., over 3 months) typically results in no marked liver fibrogenesis in mice.27 However, in mice with AZD1152-HQPA concentration a disruption

of liver Vhl, alcohol treatment caused marked fibrosis, compared with littermate controls treated with alcohol (Fig. 6D). The double disruption of Vhl and Hif-2α (VhlF/FHif2aF/F;AlbERcre+tamoxifen) ameliorated the increase in SMA, whereas a significant increase in SMA expression was observed in mice with a double disruption of Vhl and Hif-1α (VhlF/FHif1aF/F;AlbERcre+ tamoxifen) (Fig. 7A). Similarly, the increase in fibrosis observed in Vhl-disrupted mice on an alcohol diet was completely lost in the Vhl and Hif-2α double knockout,

but not the Vhl and Hif-1α double knockout (Fig. 7B). Consistent with the role of HIF-2α in exacerbating fibrosis, fibrogenic gene-expression levels were not increased in the Vhl and Hif-2α knockout, as compared to mice with 上海皓元 a Vhl disruption (Fig. 7C). Together, these data demonstrate that HIF-2α is a critical transcription factor in exacerbating fibrosis in the liver. To assess whether HIF-2α could directly regulate fibrogenic genes in the liver, ChIP assays were performed using cross-linked liver DNA isolated from tamoxifen-treated VhlF/F and VhlF/F;AlbERcre mice, with the average shearing size of 1.5 kb. Primers were designed to the center of the proximal promoter to assess HIF-2α occupancy. This method provides an assessment of HIF-2α occupancy at promoters without defining the precise HIF response element. With this method, it was shown that HIF-2α was enriched at the promoters of several fibrogenic genes in VhlF/F;AlbERcre mice, compared with control littermates (Fig. 8A).

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