Mitochondrial uncouplers inhibit hepatic stellate cell activation.
BACKGROUND
Mitochondrial dysfunction participates in the progression of several pathologies. Although there is increasing evidence for a mitochondrial role in liver disease, little is known about its contribution to hepatic stellate cell (HSC) activation. In this study we investigated the role of mitochondrial activity through mild uncoupling during in vitro activation of HSCs.


METHODS
Cultured primary human and mouse HSCs were treated with the chemical uncouplers FCCP and Valinomycin. ATP levels were measured by luciferase assay and production of reactive oxygen species was determined using the fluorescent probe DCFH-DA. Possible cytotoxicity by uncoupler treatment was evaluated by caspase 3/7 activity and cytoplasmic protease leakage. Activation of HSCs and their response to the pro-fibrogenic cytokine TGF-β was evaluated by gene expression of activation markers and signal mediators using RT-qPCR. Proliferation was measured by incorporation of EdU and protein expression of α-smooth muscle actin was analyzed by immunocytochemistry and western blot.


RESULTS
FCCP and Valinomycin treatment mildly decreased ATP and reactive oxygen species levels. Both uncouplers increased the expression of mitochondrial genes such as Tfam and COXIV while inducing morphological features of quiescent mouse HSCs and abrogating TGF-β signal transduction. Mild uncoupling reduced HSC proliferation and expression of pro-fibrogenic markers of mouse and human HSCs.


CONCLUSIONS
Mild mitochondrial uncoupling inhibits culture-induced HSC activation and their response to pro-fibrogenic cytokines like TGF-β. These results therefore suggest mitochondrial uncoupling of HSCs as a strategy to reduce progression of liver fibrosis.
Department of Cell Biology, Liver Cell Biology Lab, Vrije Universiteit Brussel, Laarbeeklaan 103, Brussels 1090, Belgium.
Eduardo L
EL
Guimarães
Jan
J
Best
Laurent
L
Dollé
Mustapha
M
Najimi
Etienne
E
Sokal
Leo A
LA
van Grunsven
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000502:physiology; D023421:Models, Animal; D017382:Reactive Oxygen Species / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology*; D014634:Valinomycin / Q000494:pharmacology*
D000199:Actins; D017382:Reactive Oxygen Species; D014475:Uncoupling Agents; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D053148:Caspase 3; D053179:Caspase 7