PTU-053 The effect of hypoxic stress on activated hepatic stellate cell chemotaxis in-vitro
Introduction Hepatic stellate cells (HSC) are believed to be the most important precursor of matrix-producing myofibroblasts that promote fibrosis following liver injury. In their activated form, HSCs migrate towards inflammatory foci in response to chemokines. When exposed to certain stresses, liver cells can express chemokines that control HSC migration. The aim of this study was to investigate the effect of hypoxia on chemokine expression by non-parenchymal liver cells using an in vitro model of ischaemia.
Methods Human HSCs, biliary epithelial cells (BECs) and Kupffer cells (KC) were isolated from normal-appearing liver tissue obtained from liver resection specimens (n=3). HSCs were cultured on either gel matrix or uncoated plastic in order to investigate both quiescent (qHSC) and activated (aHSC) phenotypes. Conditioned medium from each cell type was collected at various time points in culture after exposure to either normoxia (21% O2) or hypoxia (1% O2). For migration assays, fully activated human HSCs were used and were seeded on cell culture inserts exposed to conditioned medium from each cell type added to the lower compartment. Cells were then incubated for 20 h following which the migrated cells were fixed, stained and counted.
Results HSCs demonstrated greater migration towards medium obtained from HSCs cultured for 3 days under hypoxic conditions compared to normoxic HSCs (p=0.06). This effect decreased with longer culture times, reaching levels significantly lower than baseline by day 7 for both groups (p<0.0001). In contrast, medium obtained from HSCs cultured on gel matrix under normoxic conditions stimulated significantly higher HSC migration compared to hypoxic qHSCs, with a peak effect by day 5 (p<0.0001). Similarly, normoxic KCs stimulated significantly enhanced HSCs migration compared to hypoxic KCs (p<0.0001). On the other hand, hypoxic BECs attracted significantly more HSCs on day 1 compared to normoxia, an effect that continued to rise on day 7 (p=0.002).
Conclusion This study demonstrates that hypoxia in an in vitro model stimulates contrasting responses depending on cell type and activation state. KCs and qHSCs in a relatively high O2 state similar to that of reperfusion during liver surgery can promote chemotaxis of aHSCs possibly through the formation of reactive oxygen species. BECs and aHSCs appear to produce factors that negatively affect HSC chemotaxis as evident by below-baseline migration responses. Hypoxia attenuates this negative effect in vitro.
Competing interests None declared.