Reactive gamma-ketoaldehydes as novel activators of hepatic stellate cells in vitro

Products of lipid oxidation, such as 4-hydroxynonenal (4-HNE), are key activators of hepatic stellate cells (HSC) to a pro-fibrogenic phenotype. Isolevuglandins (IsoLG) are a family of acyclic γ-ketoaldehydes formed through oxidation of arachidonic acid or as by-products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at forming protein adducts and cross-links at concentrations 100-fold lower than 4-hydroxynonenal. Since the contribution of IsoLGs to liver injury has not been studied, we synthesized 15-E₂-IsoLG and used it to investigate whether IsoLG could induce activation of HSC.

Primary human HSC were exposed to 15-E₂-IsoLG for up to 48h. Exposure to 5μM 15-E₂-IsoLG in HSCs promoted cytotoxicity and apoptosis. At non-cytotoxic doses (50 pM-500nM) 15-E₂-IsoLG promoted HSC activation, indicated by increased expression of α-SMA, sustained activation of ERK and JNK signaling pathways, and increased mRNA and/or protein expression of cytokines and chemokines, which was blocked by inhibitors of JNK and NF-kB. In addition, IsoLG promoted formation of reactive oxygen species, and induced an early activation of ER stress, followed by autophagy. Inhibition of autophagy partially reduced the pro-inflammatory effects of IsoLG, suggesting that it might serve as a cytoprotective response.

This study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis.

IsoLGs represent a newly identified class of activators of HSC in vitro, which are biologically active at concentrations as low as 500 pM, and are particularly effective at promoting a pro-inflammatory response and autophagy.