The novel cyclophilin inhibitor C105SR reduces hepatic ischaemia-reperfusion injury via mitoprotection.

Background & Aims: Mitochondrial permeability transition pore (mPTP) opening is critical for mediating cell death during hepatic ischaemia-reperfusion injury (IRI). Blocking mPTP opening by inhibiting cyclophilin D (CypD) is a promising pharmacological approach for the treatment of IRI. Here, we show that diastereoisomers of a new class of small-molecule cyclophilin inhibitors (SMCypIs) have properties that make them attractive candidates for the development of therapeutic agents against liver IRI. Methods: Derivatives of the parent SMCypI were synthesised and evaluated for their ability to inhibit CypD peptidyl-prolyl cis-trans isomerase (PPIase) activity and for their mitoprotective properties, evaluated by measuring mitochondrial swelling and calcium retention capacity in liver mitochondria. The ability of the selected compounds to inhibit mPTP opening was evaluated in cells subjected to hypoxia/reoxygenation using a calcein/cobalt assay. Their ability to inhibit cell death was evaluated in cells subjected to hypoxia/reoxygenation by measuring lactate dehydrogenase (LDH) release, propidium iodide staining, and cell viability. The compound performing best in vitro was selected for in vivo efficacy evaluation in a mouse model of hepatic IRI. Results: The two compounds that showed the strongest inhibition of CypD PPIase activity and mPTP opening, C105 and C110, were selected. Their SR diastereoisomers carried the activity of the racemic mixture and exhibited mitoprotective properties superior to those of the known macrocyclic cyclophilin inhibitors cyclosporin A and alisporivir. C105SR was more potent than C110SR in inhibiting mPTP opening and prevented cell death in a model of hypoxia/reoxygenation. Finally, C105SR substantially protected against hepatic IRI in vivo by reducing hepatocyte necrosis and apoptosis. Conclusions: We identified a novel cyclophilin inhibitor with strong mitoprotective properties both in vitro and in vivo that represents a promising candidate for cellular protection in hepatic IRI. Impact and Implications: Hepatic ischaemia-reperfusion injury (IRI) is one of the main causes of morbidity and mortality during or after liver surgery. However, no effective therapies are available to prevent or treat this devastating syndrome. An attractive strategy to prevent hepatic IRI aims at reducing cell death by targeting mitochondrial permeability transition pore opening, a phenomenon regulated by cyclophilin D. Here, we identified a new small-molecule cyclophilin inhibitor, and demonstrated the enhanced mitoprotective and hepatoprotective properties of one of its diastereoisomers both in vitro and in vivo, making it an attractive lead compound for subsequent clinical development.

Macrophage autophagy protects against hepatocellular carcinogenesis in mice.

Autophagy is a lysosomal degradation pathway of cellular components that regulates macrophage properties. Macrophages are critically involved in tumor growth, metastasis, angiogenesis and immune suppression. Here, we investigated whether macrophage autophagy may protect against hepatocellular carcinoma (HCC). Experiments were performed in mice with deletion of the autophagy gene Atg5 in the myeloid lineage (ATG5Mye-/- mice) and their wild-type (WT) littermates. As compared to WT, ATG5Mye-/- mice were more susceptible to diethylnitrosamine (DEN)-induced hepatocarcinogenesis, as shown by enhanced tumor number and volume. Moreover, DEN-treated ATG5Mye-/- mice exhibited compromised immune cell recruitment and activation in the liver, suggesting that macrophage autophagy invalidation altered the antitumoral immune response. RNA sequencing showed that autophagy-deficient macrophages sorted from DEN mice are characterized by an enhanced expression of immunosuppressive markers. In vitro studies demonstrated that hepatoma cells impair the autophagy flux of macrophages and stimulate their expression of programmed cell death-ligand 1 (PD-L1), a major regulator of the immune checkpoint. Moreover, pharmacological activation of autophagy reduces hepatoma cell-induced PD-L1 expression in cultured macrophages while inhibition of autophagy further increases PD-L1 expression suggesting that autophagy invalidation in macrophages induces an immunosuppressive phenotype. These results uncover macrophage autophagy as a novel protective pathway regulating liver carcinogenesis.

Small-Molecule Inhibitors of Cyclophilins Block Opening of the Mitochondrial Permeability Transition Pore and Protect Mice From Hepatic Ischemia/Reperfusion Injury.

BACKGROUND & AIMS: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury. METHODS: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif-/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis. RESULTS: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 µmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 µmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif-/- mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. CONCLUSIONS: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.

The Cannabinoid Receptor 2 Protects Against Alcoholic Liver Disease Via a Macrophage Autophagy-Dependent Pathway.

Kupffer cells, the resident macrophages of the liver, play a major role in the pathogenesis of alcoholic liver disease. We have previously demonstrated that CB2 receptor protects against alcoholic liver disease by inhibiting alcohol-induced inflammation and steatosis via the regulation of Kupffer cell activation. Here, we explored the mechanism underlying these effects and hypothesized that the anti-inflammatory properties of CB2 receptor in Kupffer cells rely on activation of autophagy. For this purpose, mice invalidated for CB2 receptor (CB2(Mye-/-) mice) or for the autophagy gene ATG5 (ATG5(Mye-/-) mice) in the myeloid lineage, and their littermate wild-type mice were subjected to chronic-plus-binge ethanol feeding. CB2(Mye-/-) mice showed exacerbated alcohol-induced pro-inflammatory gene expression and steatosis. Studies in cultured macrophages demonstrated that CB2 receptor activation by JWH-133 stimulated autophagy via a heme oxygenase-1 dependent pathway. Moreover, JWH-133 reduced the induction of inflammatory genes by lipopolysaccharide in wild-type macrophages, but not in ATG5-deficient cells. The CB2 agonist also protected from alcohol-induced liver inflammation and steatosis in wild-type mice, but not in ATG5(Mye-/-) mice demonstrating that macrophage autophagy mediates the anti-inflammatory and anti-steatogenic effects of CB2 receptor. Altogether these results demonstrate that CB2 receptor activation in macrophages protects from alcohol-induced steatosis by inhibiting hepatic inflammation through an autophagy-dependent pathway.

Macrophage autophagy protects against liver fibrosis in mice.

Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5(fl/fl) LysM-Cre mice, referred to as atg5(-/-)) and their wild-type (Atg5(fl/fl), referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5(-/-) mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5(-/-) mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5(-/-) mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5(-/-) mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5(-/-) macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5(-/-) mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.

Aryl hydrocarbon receptor-dependent induction of liver fibrosis by dioxin.

The contribution of environmental pollutants to liver fibrosis is an important and poorly explored issue. In vitro studies suggest that the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands induce several genes that are known to be upregulated during liver fibrosis. Our aim was to determine whether exposure to such pollutants can lead to liver fibrosis and to characterize the mechanisms of action. Mice were treated for 2, 14, or 42 days, once a week with 25 µg/kg of TCDD. Gene and protein expression, in vitro and in vivo, as well as liver histology were investigated for each treatment. Treatment of mice with TCDD for 2 weeks modified the hepatic expression of markers of fibrosis such as collagen 1A1 and α-smooth muscle actin. This is not observed in AhR knockout mice. Following 6 weeks of treatment, histological features of murine hepatic fibrosis became apparent. In parallel, the levels of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor α) and of markers of activated fibroblasts(fibroblast-specific protein 1) were found to be upregulated. Interestingly, we also found increased expression of genes of the TGF-ß pathway and a concomitant decrease of miR-200a levels. Because the transcription factors of the Snail family were shown to be involved in liver fibrosis, we studied their regulation by TCDD. Two members of the Snail family were increased, whereas their negative targets, the epithelial marker E-cadherin and Claudin 1, were decreased. Further, the expression of mesenchymal markers was increased. Finally, we confirmed that Snai2 is a direct transcriptional target of TCDD in the human hepatocarcinoma cell line, HepG2. The AhR ligand, TCDD, induces hepatic fibrosis by directly regulating profibrotic pathways.

Beneficial paracrine effects of cannabinoid receptor 2 on liver injury and regeneration.

UNLABELLED: The cannabinoid receptor 2 (CB2) plays a pleiotropic role in innate immunity and is a crucial mediator of liver disease. In this study, we investigated the impact of CB2 receptors on the regenerative process associated with liver injury. Following acute hepatitis induced by carbon tetrachloride (CCl(4)), CB2 was induced in the nonparenchymal cell fraction and remained undetectable in hepatocytes. Administration of CCl(4) to CB2(-/-) mice accelerated liver injury, as shown by increased alanine/aspartate aminotransferase levels and hepatocyte apoptosis, and delayed liver regeneration, as reflected by a retarded induction of hepatocyte proliferating cell nuclear antigen expression; proliferating cell nuclear antigen induction was also delayed in CB2(-/-) mice undergoing partial hepatectomy. Conversely, following treatment with the CB2 agonist JWH-133, CCl(4)-treated WT mice displayed reduced liver injury and accelerated liver regeneration. The CCl(4)-treated CB2(-/-) mice showed a decrease in inducible nitric oxide synthase and tumor necrosis factor-alpha expression, and administration of the nitric oxide donor moldomine (SIN-1) to these animals reduced hepatocyte apoptosis, without affecting liver regeneration. Impaired liver regeneration was consecutive to an interleukin-6 (IL-6)-mediated decrease in matrix metalloproteinase 2 (MMP-2) activity. Indeed, CCl(4)-treated CB2(-/-) mice displayed lower levels of hepatic IL-6 messenger RNA and increased MMP-2 activity. Administration of IL-6 to these mice decreased MMP-2 activity and improved liver regeneration, without affecting hepatocyte apoptosis. Accordingly, administration of the MMP inhibitor CTTHWGFTLC to CCl(4)-treated CB2(-/-) mice improved liver regeneration. Finally, in vitro studies demonstrated that incubation of hepatic myofibroblasts with JWH-133 increased tumor necrosis factor-alpha and IL-6 and decreased MMP-2 expressions. CONCLUSION: CB2 receptors reduce liver injury and promote liver regeneration following acute insult, via distinct paracrine mechanisms involving hepatic myofibroblasts. These results suggest that CB2 agonists display potent hepatoprotective properties, in addition to their antifibrogenic effects.

The sphingosine 1-phosphate receptor S1P2 triggers hepatic wound healing.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid produced by sphingosine kinase (SphK1 and 2). We previously showed that S1P receptors (S1P1, S1P2, and S1P3) are expressed in hepatic myofibroblasts (hMF), a population of cells that triggers matrix remodeling during liver injury. Here we investigated the function of these receptors in the wound healing response to acute liver injury elicited by carbon tetrachloride, a process that associates hepatocyte proliferation and matrix remodeling. Acute liver injury was associated with the induction of S1P2, S1P3, SphK1, and SphK2 mRNAs and increased SphK activity, with no change in S1P1 expression. Necrosis, inflammation, and hepatocyte regeneration were similar in S1P2-/- and wild-type (WT) mice. However, compared with WT mice, S1P2-/- mice displayed reduced accumulation of hMF, as shown by lower induction of smooth muscle alpha-actin mRNA and lower induction of TIMP-1, TGF-beta1, and PDGF-BB mRNAs, overall reflecting reduced activation of remodeling in response to liver injury. The wound healing response was similar in S1P3-/- and WT mice. In vitro, S1P enhanced proliferation of cultured WT hMF, and PDGF-BB further enhanced the mitogenic effect of S1P. In keeping with these findings, PDGF-BB up-regulated S1P2 and SphK1 mRNAs, increased SphK activity, and S1P2 induced PDGF-BB mRNA. These effects were blunted in S1P2-/- cells, and S1P2-/- hMF exhibited reduced mitogenic and comitogenic responses to S1P. These results unravel a novel major role of S1P2 in the wound healing response to acute liver injury by a mechanism involving enhanced proliferation of hMF.

Antifibrogenic role of the cannabinoid receptor CB2 in the liver.

BACKGROUND & AIMS: Hepatic myofibroblasts are central for the development of liver fibrosis associated with chronic liver diseases, and blocking their accumulation may prevent fibrogenesis. Cannabinoids are the active components of marijuana and act via 2 G-protein-coupled receptors, CB1 and CB2. Here, we investigated whether liver fibrogenic cells are a target of cannabinoids. METHODS: CB2 receptors were characterized in biopsy specimens of normal human liver and active cirrhosis by immunohistochemistry, and in cultures of hepatic stellate cells and hepatic myofibroblasts by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and GTPgammaS assays. Functional studies were performed in cultured hepatic myofibroblasts and activated hepatic stellate cells. Carbon tetrachloride-induced liver fibrosis was studied in mice invalidated for CB2 receptors. RESULTS: In liver biopsy specimens from patients with active cirrhosis of various etiologies, CB2 receptors were expressed in nonparenchymal cells located within and at the edge of fibrous septa in smooth muscle alpha-actin-positive cells. In contrast, CB2 receptors were not detected in normal human liver. CB2 receptors were also detected in cultured hepatic myofibroblasts and in activated hepatic stellate cells. Their activation triggered potent antifibrogenic effects, namely, growth inhibition and apoptosis. Growth inhibition involved cyclooxygenase-2, and apoptosis resulted from oxidative stress. Finally, mice invalidated for CB2 receptors developed enhanced liver fibrosis following chronic carbon tetrachloride treatment as compared with wild-type mice. CONCLUSIONS: These data constitute the first demonstration that CB2 receptors are highly up-regulated in the cirrhotic liver, predominantly in hepatic fibrogenic cells. Moreover, this study also highlights the antifibrogenic role of CB2 receptors during chronic liver injury.

Hepatic fibrosis: molecular mechanisms and drug targets.

Liver fibrosis is the common response to chronic liver injury, ultimately leading to cirrhosis and its complications, portal hypertension, liver failure, and hepatocellular carcinoma. Efficient and well-tolerated antifibrotic drugs are currently lacking, and current treatment of hepatic fibrosis is limited to withdrawal of the noxious agent. Efforts over the past decade have mainly focused on fibrogenic cells generating the scarring response, although promising data on inhibition of parenchymal injury and/or reduction of liver inflammation have also been obtained. A large number of approaches have been validated in culture studies and in animal models, and several clinical trials are underway or anticipated for a growing number of molecules. This review highlights recent advances in the molecular mechanisms of liver fibrosis and discusses mechanistically based strategies that have recently emerged.

Molecular mechanisms regulating the antifibrogenic protein heme-oxygenase-1 in human hepatic myofibroblasts.

BACKGROUND/AIMS: Hepatic myofibroblasts are central in liver fibrogenesis associated with chronic liver diseases. We previously showed that heme-oxygenase-1 (HO-1) displays antifibrogenic properties in human hepatic myofibroblasts. Here, we further investigated the mechanisms regulating HO-1 expression. METHODS: Expression of HO-1 was assayed in cultured human hepatic myofibroblasts by Northern and Western blot. Functional studies were also performed in cultured human hepatic myofibroblasts. RESULTS: 15-Deoxy-Delta(12,14)-prostaglandin J2 (15-d-PGJ2) elicited inhibition of proliferation and of alpha1(I) collagen mRNA expression. These effects were reproduced by the glutathione depletor diethyl maleate and blunted by the glutathione precursor N-acetyl cysteine, indicating the involvement of oxidative stress. Two consecutive events mediated inhibition of proliferation and of alpha1(I) collagen mRNA expression by 15-d-PGJ2: (i) mild oxidative stress characterized by a transient GSH decrease and (ii) activation of p38 MAPK, resulting in increased HO-1 mRNA stability. CONCLUSIONS: Our results provide new insights into the regulatory mechanisms governing HO-1 expression in human hepatic myofibroblasts and identify mild oxidative stress and p38 MAPK as two consecutive early signals promoting HO-1 induction that are crucial for its antifibrogenic properties, namely inhibition of growth and extracellular matrix gene expression.

A cysteine-linkable, short cleavable photoprobe with dual functionality to explore protein-protein interfaces.

We developed a bifunctional photoprobe with dual functionality, that can be specifically tethered to cysteinyl residues of peptides and proteins through a short cleavable disulfide bond. Thus, an aryldiazonium moiety is positioned at approximately 8.5 A from the modified cysteinyl alpha-carbon, leading to one of the shortest cleavable linkages. In a sodium azide-containing buffer, the aryldiazonium moiety is transformed into an aryl azide. Therefore, with one bifunctional photoprobe two types of photogenerated species can be obtained: a hydrophilic and positively charged arylcation or a hydrophobic nitrene. We coupled the aryldiazonium probe, in a site-directed manner, to a nicotinic acetylcholine receptor competitive antagonist, obtained by chemical engineering of an analogue of a snake alpha-neurotoxin. In this molecule, Arg33, which is known to interact with the receptor, was replaced by a cysteine residue, where the photoprobe could be attached. Under inactinic light, this novel photosensitive snake toxin behaved as a reversible ligand on the Torpedo acetylcholine receptor. However, when irradiated at 391 nm, it generated a highly reactive arylcation which labeled mostly the receptor alpha-subunit, confirming the location of the tip of the second toxic loop near this receptor subunit. Finally, we showed that reduction of the disulfide bond, linking the ligand to the photocoupled receptor, allowed introduction of radioactivity on the labeled residue(s), opening the way to further characterization and avoiding the synthesis of a radioactive bifunctional photoprobe.