The endogenous cannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces apoptosis in many cell types. Although AEA levels are elevated in liver fibrosis, its role in fibrogenesis remains unclear. This study investigated effects of AEA in primary hepatic stellate cells (HSCs). Anandamide blocked HSC proliferation at concentrations of 1 to 10 mol/L but did not affect HSC proliferation or activation at nanomolar concentrations. At higher concentrations (25-100 mol/L), AEA rapidly and dose-dependently induced cell death in primary culture-activated and in vivo-activated HSCs, with over 70% cell death after 4 hours at 25 mol/L. In contrast to treatment with Fas ligand or gliotoxin, AEA-mediated death was caspase independent and showed typical features of necrosis such as rapid adenosine triphosphate depletion and propidium iodide uptake. Anandamide-induced reactive oxygen species (ROS) formation, and an increase in intracellular Ca 2؉ . Pretreatment with the antioxidant glutathione or Ca 2؉ -chelation attenuated AEA-induced cell death. Although the putative endocannabinoid receptors CB1, CB2, and VR1 were expressed in HSCs, specific receptor blockade failed to block cell death. Depletion of membrane cholesterol by methyl--cyclodextrin inhibited AEA binding, blocked ROS formation and intracellular Ca 2؉ -increase, and prevented cell death. In primary hepatocytes, AEA showed significantly lower binding and failed to induce cell death even after prolonged treatment. In conclusion, AEA efficiently induces necrosis in activated HSCs, an effect that depends on membrane cholesterol and a subsequent increase in intracellular Ca 2؉ and ROS. The anti-proliferative effects and the selective killing of HSCs, but not hepatocytes, indicate that AEA may be used as a potential anti-fibrogenic tool. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/ 0270-9139/suppmat/index.html). (HEPATOLOGY 2005;41:1085-1095.)
See Editorial on Page 983 L iver fibrosis is a common response to chronic hepatic damage induced by a variety of insults, including viral and parasitic infection, drugs, alcohol abuse, iron and copper overload, nonalcoholic steatohepatitis, and autoimmune diseases. Hepatic stellate cells (HSCs) are believed to play a key role in the development and resolution of liver fibrosis. In the injured liver, HSCs undergo an activation process that results in a phenotypic change from retinoid-storing quiescent cells to activated, collagen-producing HSCs with a myofibroblast phenotype. 1 The resolution of liver fibrosis correlates with an increase in HSC apoptosis, 2,3 and current treatment strategies for liver fibrosis include the induction of cell death in HSCs. 4 In particular, members of the tumor necrosis factor (TNF) receptor-family including TNF␣, TNF-related apoptosis-inducing ligand, nerve growth factor, and Fas ligand (FasL), have been shown to induce apoptosis in HSCs. [5][6][7][8] Moreover, gliotoxin, a toxin produced by Aspergillus fumigatus, causes apoptosis in HSCs. 9 Anandamide (N-arachidonoylethanolamine; AEA) is the main endogenous agonist among a recently discov-