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

Abstract

Aims. 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 -ketoaldehydes formed through oxidation of arachidonic acid or as by-products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at Longato 2 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-E 2 -IsoLG and used it to investigate whether IsoLG could induce activation of HSC. Results. Primary human HSC were exposed to 15-E 2 -IsoLG for up to 48 hours.Exposure to 5 M 15-E 2 -IsoLG in HSCs promoted cytotoxicity and apoptosis. At non-cytotoxic doses (50 pM-500 nM) 15-E 2 -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. Innovation. This study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis. Conclusions. 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.

MAIT cells are chronically activated in patients with autoimmune liver disease and promote profibrogenic hepatic stellate cell activation

“…Liver‐associated lymphocytes were isolated from cirrhotic liver tissue derived from explanted livers of patients with AILD, from wedge sections of patients undergoing liver surgery in the absence of background liver disease, or from healthy liver explants considered unsuitable for transplantation (enabled by NHSBT). Primary human HSCs (hHSCs) were isolated from wedge sections of human liver tissue as described previously …”

“…Primary human HSCs (hHSCs) were isolated from wedge sections of human liver tissue as described previously. (19) FLOW CYTOMETRIC ANALYSIS Cells were stained for surface markers and intracellular markers (see Supporting Information for a full list of antibodies). Dead cells were excluded using a live/ dead fixable UV dead cell stain kit (Invitrogen, Carlsbad, CA).…”

The adenosine monophosphate–activated protein kinase—vacuolar adenosine triphosphatase–pH axis: A key regulator of the profibrogenic phenotype of human hepatic stellate cells

“…Primary hHSCs were cultured in 20% serum‐enriched medium for 24 hours, followed by incubation in serum‐free medium (SFM) for 24 hours before each experimental treatment to avoid interference with growth factors and amino acids enriched in fetal bovine serum . Data obtained from cells cultured in complete medium (CM) are reported in figure legends.…”

“…(15) TREATMENTS Primary hHSCs were cultured in 20% serumenriched medium for 24 hours, followed by incubation in serum-free medium (SFM) for 24 hours before each experimental treatment to avoid interference with growth factors and amino acids enriched in fetal bovine serum. (16)(17)(18) Data obtained from cells cultured in complete medium (CM) are reported in figure legends. Specific treatment with the v-ATPase inhibitors bafilomycin A1 and KM91104; with the AMPK activators diflunisal, A769662, and ZLN024 hydrochloride; and with TGFb1 and PDGF-BB are described in the Supporting Information.…”

Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds

“…The mechanisms of protein crossing-linking by isolevuglandins have been investigated, and found to occur both by aminal and pyrrole-pyrrole cross-links [72]. Treatment of hepatic stellate cells with isolevuglandins caused their activation, and was thought to depend on formation of protein adducts, although this was not explicitly demonstrated [73].…”