The novel endocannabinoid virodhamine selectively induces cell death in hepatic stellate cells but not in hepatocytes

Aims: We have previously shown that the endogenous cannabinoid anandamide (N-arachidonoyl ethanolamide, AEA) is an antifibrotic lipid mediator that blocks proliferation and induces cell death in hepatic stellate cells (HSCs), but not in hepatocytes. However, the effects on hepatic cell populations of the novel endocannabinoid virodhamine (O-arachidonoyl ethanolamine), whose tissue levels exceed those of AEA >9 times, has not been investigated so far. Methods: Primary rat hepatocytes and HSCs were isolated by collagenase perfusion. AEA- and virodhamine-induced cell death was analysed by LDH assay and annexin V/propidium iodide staining. Reactive oxygen species (ROS) formation was monitored by DCFDA fluorescence. Results: Similarly to AEA, virodhamine dose-dependently induced cell death in HSCs after 2–4h, starting from 5µM. As AEA, virodhamine induced necrotic cell death in HSCs (positive propidium iodide, lack of annexin V staining). Virodhamine induced massive ROS formation (>75% compared to AEA). The antioxidant tocopherol significantly decreased virodhamine death, demonstrating an ROS-dependent mechanism. Cannabinoid receptor antagonists for CB1 and CB2 failed to block virodhamine death, indicating a receptor-independent mechanism. Similar to AEA, hepatocytes were resistant to virodhamine due to high levels of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), since FAAH inhibition with URB597 significantly increased virodhamine-induced cell death in hepatocytes. Accordingly, adenoviral FAAH overexpression in HSCs lead to complete resistance against virodhamine. Conclusion: Virodhamine efficiently induced necrotic cell death in activated HSCs, but not in hepatocytes. Virodhamine-caused cell death was mediated by pronounced ROS formation, but not through engagement of cannabinoid receptors. The selective induction of cell death in HSCs and its high tissue levels proposes virodhamine as a potential endogenous antifibrogenic mediator.

Hepatic stellate cells - antifibrotic - cell death - endocannabinoids - hepatocytes - virodhamine