Hepatic iron overload in alcoholic liver disease: The role of sinusoidal endothelial cells in iron sensing

Background and Aims:

So far, hepatic iron overload in patients with alcoholic liver disease is poorly understood. Hepcidin, the master switch of systemic iron homeostasis and is regulated by the BMP signaling pathway. Recent data showed that liver sinusoidal endothelial cells (LSECs) express the highest amount of BMP6 among different hepatic cell types and are able to regulate iron homeostasis in vivo. However, the exact mechanisms, how iron levels are sensed by ECs and how BMP signaling is involved in the regulation of systemic iron metabolism as well as which cells are involved is still not completely known. The aim of this study is to investigate the crosstalk between LSECs and hepatocytes in regulating iron metabolism.

Methods:

Huh7 cells (hepatocytes) and HUVECs (Human Umbilical Vein Endothelial Cells) were cultured alone and treated with holo-transferrin (Holo-Tf), ferric ammonium citrate (FAC) and salicylaldehyde isonicotinoyl hydrazine (SIH) as an iron chelator under hypoxic condition (1% O2) for 24 hours. Next, co-cultures of HUVECs and Huh7 cells were established by using the supernatant of HUVECs to incubate Huh7 cells. Hepcidin, BMP6, TFR1 were assessed by qRT-PCR or Western blot. Meanwhile the Bmp6 concentrations in medium were detected by ELISA. To investigate the function of BMP6 in the co-culture system, we also neutralized BMP6 with monoclonal antibody in the supernatant of HUVECs and incubated Huh7 cells with the neutralized supernatant.

Results:

Ferric iron significantly led HUVECs to secret more Bmp6 into the culturing supernatant under 1% O2, whereas no effect on Huh7 cells was detected. Expression of hepcidin in Huh7 cells was remarkably increased after incubation with the supernatant of HUVECs cultured under 1% O2 for 24h regardless of treatments with iron if compared to fresh medium control group. In the meantime, hepcidin expression in Huh7 cells induced by the supernatant of HUVECs could be remarkably blocked by using neutralizing BMP6 antibody.

Conclusion:

Endothelial cells are able to sense iron changes (iron supplementation or chelation) and the production of endothelial cells can induce the hepcidin expression in hepatocytes. BMP6 played an essential role in hepcidin expression in hepatocytes, whereas the hepcidin expression quantity is not related to the BMP6 concentration in the surroundings. Further explorations are necessary to better understand the crosstalk between endothelial cells and hepatocytes on iron regulation.