Role of the Hypoxia Inducible Factors HIF-1α and HIF-2α during hepatic repair in a rat model of thioacetamide (TAA)-induced chronic liver damage

Background: Hypoxia has been shown to have a role in the pathogenesis of several forms of liver disease. The Hypoxia Inducible Factors (HIFs) are a family of evolutionarily conserved transcriptional regulators that affect a homeostatic response to low oxygen tension and have been identified as key mediators of angiogenesis, inflammation, and metabolism. HIF-1α and HIF-2α can activate simultaneously distinct target genes with different functions regarding the regulation of hypoxia-related genes.

Methods: Male Wistar rats were administrated thioacetamide (TAA) in drinking water up to 20 weeks and sacrificed at different time points (4, 8, 12, 16, 20 weeks). Expression of HIF-1α and HIF-2α was evaluated during liver damage and cholangiocarcinoma development after TAA administration. Expression of HIF-1α and HIF-2α 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: The immunofluorescence staining with anti-HIF-1α identified a co-localization with α-SMA, a hepatic stellate cells marker, during 8 and 16 weeks of hepatic damage after TAA administration. However HIF-2α appeared in α-SMA positive cells during late time point of the study (16 weeks). HIF-2α was localized mainly in proximity with the sinusoidal spaces and in the endothelial cells, showed positivity in ED1 cells during early time of study (8w) while HIF-1α positivity in ED1 cells was detected during late time points (16w). Immunofluorescence analysis of liver from TAA administrated rat revealed HIF-2α strong positivity in ED2 cells while ED2 cells were negative for HIF-1α. RT-PCR and Western Blot data confirmed the immunohistochemical analysis as they showed a sharp up-regulation of HIF-1α reaching the highest level 8 weeks after beginning the experiment whereas HIF-2α showed a maximum increase of gene expression at 16 weeks.

Conclusion: HIF-1α expression in the hypoxic liver area during early time point after TAA-administration and its reduction during the late time point clearly describes its role as damage-associated molecule during hepatic repair and fibrogenesis. The detection of HIF-2α at a late time point (16w) reveals that under hypoxic conditions a decline of HIF-1α in favor of HIF-2α-dependent gene regulation takes place and indicates a role of HIF-2α during the recovery phase of a chronic liver damage.

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 ED2+ cells opens a new scenario in the role of the macrophage compartment in the process of erythropoiesis.