Superoxide-induced apoptosis of activated rat hepatic stellate cells.
OBJECTIVE
During liver injury, reactive oxygen species (ROS) are produced by the resident macrophages (Kupffer cells) and infiltrating blood cells such as neutrophils. ROS cause transformation of desmin-positive quiescent hepatic stellate cells (HSCs) into the proliferating activated phenotype that expresses alpha-smooth muscle actin (alpha-SMA). The highly fibrogenic and contractile activated HSCs (aHSCs) produce various cytokines and growth factors, and play important role in the pathophysiology of chronic liver disease. However, apoptotic aHSCs are also observed during active fibrogenesis in the injured liver. Therefore, we investigated the mechanisms of apoptosis of aHSCs in relation to ROS.


METHODS
HSCs, isolated from normal rat liver, were activated in culture and effects of superoxide were determined between subcultures 3 and 5.


RESULTS
Treatment with superoxide caused apoptosis of aHSCs as determined by flow cytometry, TUNEL assay and DNA laddering analysis. The mechanisms of superoxide-induced apoptosis involved release of cytochrome c, increased Bax expression, increased caspase-3 activity, and hydrolysis of polyADP-ribose polymerase. Superoxide also increased the expression of antiapoptotic Bcl-xL and nuclear translocation of NFkappaB. Caspase-3 inhibitor (DEVD-fmk) and antioxidants (N-acetylcysteine, vitamin E and superoxide dismutase) inhibited superoxide-induced apoptosis.


CONCLUSIONS
Superoxide-induced apoptosis of aHSCs may be a novel mechanism of limiting chronic fibrotic liver injury.
Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, E-1542 BST, 200 Lothrop street, Pittsburgh, PA 15213, USA.
Chinnasamy
C
Thirunavukkarasu
Simon
S
Watkins
Stephen A K
SA
Harvey
Chandrashekhar R
CR
Gandhi
D000818:Animals; D017209:Apoptosis; D061945:Caspase Inhibitors; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D045604:Extracellular Fluid / Q000201:enzymology; D016166:Free Radical Scavengers / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing); D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology*; D008297:Male; D010105:Oxygenases / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000494:pharmacology*
D061945:Caspase Inhibitors; D016166:Free Radical Scavengers; D006360:Heat-Shock Proteins; D011480:Protease Inhibitors; D013481:Superoxides; D010105:Oxygenases; D006419:Heme Oxygenase (Decyclizing); C491523:Hmox1 protein, rat; D013482:Superoxide Dismutase