HMGB1 inflammosome activity modulates Hypoxia inducible factor-1α Expression in a rat model of thioacetamide-induced chronic liver damage and cholangiocarcinoma development

Background: HIF-1α and HIF-2α have been identified as key mediators of angiogenesis, inflammation, and metabolism and can activate simultaneously distinct target genes with different functions regarding the regulation of hypoxia-related-genes.

Methods: Male Wistar rats and TLR-4^-/- mice were administrated thioacetamide (TAA) in drinking water up to 20 weeks and sacrificed at 4, 8, 12, 16, 20 weeks. Expression of HIF-1α and HIF-2α was evaluated during liver damage and cholangiocarcinoma. Expression of HIF-1α, HIF-2α, HMGB1 was measured by RT-PCR and Western blot. HIF-1α, HIF-2α, α-SMA, CK-19, ED1 and ED2 were localized by immunofluorescence staining.

Results: HIF-1α was colocalized with α-SMA during 8 and 16 weeks after TAA administration suggesting its role in liver fibrogenesis. However HIF-2α appeared in α-SMA-positive-cells after 16 weeks. HIF-2α was localized mainly in proximity to the sinusoidal spaces and in the endothelial cells, showed positivity in ED1-cells at 8w while HIF-1α positivity in ED1-cells was detected only 16w after treatment. Immunohistochemistry of liver from TAA-treated rat revealed HIF-2α strong positivity in ED2-cells but not for HIF-1α. RT-PCR and Western Blot data confirmed the immunohistochemical analysis showing a sharp increase of HIF-1α at 8w and of HIF-2α at 16w. Western blot analysis showed increased accumulation of HMGB1 after liver damage and cholangiocarcinoma development. In TLR-4 Knockout mice by Western blot analysis, expression of HIF-1α and HMGB1 was switched off.

Conclusion: HIF-1α expression in the hypoxic liver area during early time point after TAA-administration and its reduction during the experiment describes its role during hepatic repair and fibrogenesis. We highlight for the first time the role of HMGB1 in inducing HIF-1α gene expression during tumor development. The detection of HIF-2α at a late time point (16w) highlights a role of HIF-2α during the recovery phase. HIF-2α localization in endothelial cells during hepatic damage confirms its involvement in angiogensis and vascularization. As HIF-2α seems to have a crucial role in the induction of the hepatic erythropoietin (EPO), its co-localization in ED-2⁺ cells opens a new scenario in the role of the macrophage compartment in the process of erythropoiesis.