Hirsova, Petra

Chronic cholestatic cholangiopathies present a therapeutic challenge due to their complex pathophysiology, where the accumulation of bile acids plays a crucial role. In this study, we found that dimethyl fumarate attenuated cholestatic liver damage in a murine model through its significant anti-inflammatory and antifibrotic activity supported by reduced bile acid accumulation in the plasma and liver via their increased fecal excretion.

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300 LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300 LSECΔ/Δ mice compared to p300 fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300 LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300 LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

With the epidemic of obesity, nonalcoholic fatty liver disease (NAFLD) has become the most common pediatric liver disease. The influence of a perinatal obesity‐inducing diet (OID) on the development and progression of NAFLD in offspring is important but incompletely studied. Hence, we fed breeding pairs of C57BL/6J mice during gestation and lactation (perinatally) either chow or an OID rich in fat, fructose, and cholesterol (FFC). The offspring were weaned to either chow or an FFC diet, generating four groups: perinatal (p)Chow‐Chow, pChow‐FFC, pFFC‐Chow, and pFFC‐FFC. Mice were sacrificed at 10 weeks of age. We examined the whole‐liver transcriptome by RNA sequencing (RNA‐seq) and whole‐liver genome methylation by reduced representation bisulfite sequencing (RRBS). Our results indicated that the pFFC‐FFC mice had a significant increase in hepatic steatosis, injury, inflammation, and fibrosis, as assessed histologically and biochemically. We identified 189 genes that were differentially expressed and methylated in the pFFC‐FFC mice versus the pChow‐FFC mice. Gene set enrichment analysis identified hepatic fibrosis/hepatic stellate cell activation as the top canonical pathway, suggesting that the differential DNA methylation events in the mice exposed to the FFC diet perinatally were associated with a profibrogenic transcriptome. To verify that this finding was consistent with perinatal nutritional reprogramming of the methylome, we exposed pFFC‐Chow mice to an FFC diet in adulthood. These mice developed significant hepatic steatosis, injury, inflammation, and more importantly fibrosis when compared to the appropriate controls.Conclusion: Perinatal exposure to an OID primes the immature liver for an accentuated fibrosing nonalcoholic steatohepatitis (NASH) phenotype, likely through nutritional reprogramming of the offspring methylome. These data have potential clinical implications for monitoring children of obese mothers and risk stratification of children with NAFLD.

Abstract The Hippo pathway effector, Yes-associated protein (YAP), is a transcriptional coactivator implicated in cholangiocarcinoma (CCA) pathogenesis. YAP is known to be regulated by a serine/threonine kinase relay module (MST1/2–LATS1/2) culminating in phosphorylation of YAP at Serine 127 and cytoplasmic sequestration. However, YAP also undergoes tyrosine phosphorylation, and the role of tyrosine phosphorylation in YAP regulation remains unclear. Herein, YAP regulation by tyrosine phosphorylation was examined in human and mouse CCA cells, as well as patient-derived xenograft (PDX) models. YAP was phosphorylated on tyrosine 357 (Y357) in CCA cell lines and PDX models. SRC family kinase (SFK) inhibition with dasatinib resulted in loss of YAPY357 phosphorylation, promoted its translocation from the nucleus to the cytoplasm, and reduced YAP target gene expression, including cell lines expressing a LATS1/2-resistant YAP mutant in which all serine residues were mutated to alanine. Consistent with these observations, precluding YAPY357 phosphorylation by site-directed mutagenesis (YAPY357F) excluded YAP from the nucleus. Targeted siRNA experiments identified LCK as the SFK that most potently mediated YAPY357 phosphorylation. Likewise, inducible CRISPR/Cas9-targeted LCK deletion decreased YAPY357 phosphorylation and its nuclear localization. The importance of LCK in CCA biology was demonstrated by clinical observations suggesting LCK expression levels were associated with early tumor recurrence following resection of CCA. Finally, dasatinib displayed therapeutic efficacy in PDX models. Mol Cancer Res; 16(10); 1556–67. ©2018 AACR.

ABSTRACTSaturated fatty acids (SFA) and their toxic metabolites contribute to hepatocyte lipotoxicity in nonalcoholic steatohepatitis (NASH). We previously reported that hepatocytes, under lipotoxic stress, express the potent macrophage chemotactic ligand C‐X‐C motif chemokine 10 (CXCL10), and release CXCL10‐enriched extracellular vesicles (EV) by a mixed lineage kinase (MLK) 3‐dependent mechanism. In the current study, we sought to examine the signaling pathway responsible for CXCL10 induction during hepatocyte lipotoxicity. Here, we demonstrate a role for signal transducer and activator of transcription (STAT) 1 in regulating CXCL10 expression. Huh7 and HepG2 cells were treated with lysophosphatidylcholine (LPC), the toxic metabolite of the SFA palmitate. In LPC‐treated hepatocytes, CXCL10 induction is mediated by a mitogen activated protein kinase (MAPK) signaling cascade consisting of a relay kinase module of MLK3, MKK3/6, and p38. P38 in turn induces STAT1 Ser727 phosphorylation and CXCL10 upregulation in hepatocytes, which is reduced by genetic or pharmacological inhibition of this MAPK signaling cascade. The binding and activity of STAT1 at the CXCL10 gene promoter were identified by chromatin immunoprecipitation and luciferase gene expression assays. Promoter activation was attenuated by MLK3/STAT1 inhibition or by deletion of the consensus STAT1 binding sites within the CXCL10 promoter. In lipotoxic hepatocytes, MLK3 activates a MAPK signaling cascade, resulting in the activating phosphorylation of STAT1, and CXCL10 transcriptional upregulation. Hence, this kinase relay module and/or STAT1 inhibition may serve as a therapeutic target to reduce CXCL10 release, thereby attenuating NASH pathogenesis. J. Cell. Biochem. 118: 3249–3259, 2017. © 2017 Wiley Periodicals, Inc.

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disorder, wherein proinflammatory lipids, such as ceramide and its derivative sphingosine 1-phosphate (S1P), contribute to macrophage-associated liver inflammation. For example, we have previously demonstrated a role for S1P in steatotic hepatocyte-derived S1P-enriched extracellular vesicles in macrophage chemotaxis in vitro. Therefore, we hypothesized that FTY720, an S1P antagonist, would ameliorate NASH by inhibiting proinflammatory monocyte chemotaxis. To test our hypothesis, NASH was established in C57BL/6 male mice by feeding a diet high in fructose, saturated fat, and cholesterol for 22 wk. Then mice received daily intraperitoneal injections of FTY720 for 2 wk before analysis of liver injury, inflammation, and fibrosis. FTY720-treated mice with NASH demonstrated improved liver histology with a significant reduction in hepatocyte ballooning and inflammatory foci. Hepatomegaly was reversed, and liver triglycerides were reduced following FTY720 administration to mice with NASH. Correspondingly, serum ALT levels, hepatic inflammatory macrophage accumulation, and the expression of Ly6C in recruited myeloid cells was reduced in FTY720-treated mice. Hepatic collagen accumulation and expression of α-smooth muscle actin were significantly lowered as well. Body composition, energy consumption and utilization, and hepatic sphingolipid composition remained unchanged following FTY720 administration. FTY720 ameliorates murine nonalcoholic steatohepatitis. Reduction in liver injury and inflammation is associated with a reduction in hepatic macrophage accumulation, likely due to dampened recruitment of circulating myeloid cells into the liver. Nonalcoholic steatohepatitis may be a novel indication for the therapeutic use of FTY720. NEW & NOTEWORTHY There are no approved pharmacologic therapies for nonalcoholic steatohepatitis (NASH), the leading cause of chronic liver disease worldwide. This study describes the use of FTY720, a novel small molecule, for the amelioration of NASH in a mouse model. We demonstrate that 2-wk administration of FTY720 to mice with NASH led to a reduction in liver injury, inflammation, and fibrosis. These data provide a preclinical rationale for studying this drug in human NASH.

Extracellular vesicles (EVs) are nanometer‐sized, membrane‐bound vesicles released by cells into the extracellular milieu. EVs are now recognized to play a critical role in cell‐to‐cell communication. EVs contain important cargo in the form of proteins, lipids, and nucleic acids and serve as vectors for delivering this cargo from donor to acceptor or target cell. EVs are released under both physiologic and pathologic conditions, including liver diseases, and exert a wide range of effects on target cells. This review provides an overview on EV biogenesis, secretion, cargo, and target cell interactions in the context of select liver diseases. Specifically, the diverse roles of EVs in nonalcoholic steatohepatitis, alcoholic liver disease, viral hepatitis, cholangiopathies, and hepatobiliary malignancies are emphasized. Liver diseases often result in an increased release of EVs and/or in different cargo sorting into these EVs. Either of these alterations can drive disease pathogenesis. Given this fact, EVs represent a potential target for therapeutic intervention in liver disorders. Because altered EV composition may reflect the underlying disease condition, circulating EVs can be exploited for diagnostic and prognostic purposes as a liquid biopsy. Furthermore, ex vivo modified or synthesized EVs can be engineered as therapeutic nano‐shuttles. Finally, we highlight areas that merit further investigation relevant to understanding how EVs regulate liver disease pathogenesis. (H epatology 2016;64:2219‐2233).

3-Aminorhodanine reacts with aldehydes to form either 5-[(aryl)alkylidene]-substituted products or Schiff bases or derivatives substituted at both the 3-amino group and the 5-methylene group, depending on the reaction conditions. In this note, synthesis and characterization of 3-amino-5-(pyridin-2-ylmethylidene)-2-thioxo-1,3-thiazolidin-4-one is reported.

IQ motif–containing GTPase-activating protein 1 (IQGAP1) is a cytoskeleton-interacting scaffold protein. CXCR4 is a chemokine receptor that binds stromal cell–derived factor-1 (SDF-1; also known as CXCL12). Both IQGAP1 and CXCR4 are overexpressed in cancer cell types, yet it was unclear whether these molecules functionally interact. Here, we show that depleting IQGAP1 in Jurkat T leukemic cells reduced CXCR4 expression, disrupted trafficking of endocytosed CXCR4 via EEA-1+ endosomes, and decreased efficiency of CXCR4 recycling. SDF-1–induced cell migration and activation of extracellular signal-regulated kinases 1 and 2 (ERK) MAPK were strongly inhibited, even when forced overexpression restored CXCR4 levels. Similar results were seen in KMBC and HEK293 cells. Exploring the mechanism, we found that SDF-1 treatment induced IQGAP1 binding to α-tubulin and localization to CXCR4-containing endosomes and that CXCR4-containing EEA-1+ endosomes were abnormally located distal from the microtubule (MT)-organizing center (MTOC) in IQGAP1-deficient cells. Thus, IQGAP1 critically mediates CXCR4 cell surface expression and signaling, evidently by regulating EEA-1+ endosome interactions with MTs during CXCR4 trafficking and recycling. IQGAP1 may similarly promote CXCR4 functions in other cancer cell types.