Núria Tarrats

Critical role of tumor necrosis factor receptor 1, but not 2, in hepatic stellate cell proliferation, extracellular matrix remodeling, and liver fibrogenesis†‡§

Tumor necrosis factor (TNF) has been implicated in the progression of many chronic liver diseases leading to fibrosis; however, the role of TNF in fibrogenesis is controversial and the specific contribution of TNF receptors to hepatic stellate cell (HSC) activation remains to be established. Using HSCs from wild‐type, TNF‐receptor‐1 (TNFR1) knockout, TNF‐receptor‐2 (TNFR2) knockout, or TNFR1/R2 double‐knockout (TNFR‐DKO) mice, we show that loss of both TNF receptors reduced procollagen‐α1(I) expression, slowed down HSC proliferation, and impaired platelet‐derived growth factor (PDGF)‐induced promitogenic signaling in HSCs. TNFR‐DKO HSCs exhibited decreased AKT phosphorylation and in vitro proliferation in response to PDGF. These effects were reproduced in TNFR1 knockout, but not TNFR2 knockout, HSCs. In addition, matrix metalloproteinase 9 (MMP‐9) expression was dependent on TNF binding to TNFR1 in primary mouse HSCs. These results were validated in the human HSC cell line, LX2, using neutralizing antibodies against TNFR1 and TNFR2. Moreover, in vivo liver damage and fibrogenesis after bile‐duct ligation were reduced in TNFR‐DKO and TNFR1 knockout mice, compared to wild‐type or TNFR2 knockout mice. Conclusion: TNF regulates HSC biology through its binding to TNFR1, which is required for HSC proliferation and MMP‐9 expression. These data indicate a regulatory role for TNF in extracellular matrix remodeling and liver fibrosis, suggesting that targeting TNFR1 may be of benefit to attenuate liver fibrogenesis. (HEPATOLOGY 2011;)

Acidic sphingomyelinase controls hepatic stellate cell activation and in vivo liver fibrogenesis

The mechanisms linking hepatocellular death, hepatic stellate cell (HSC) activation, and liver fibrosis are largely unknown. Here, we investigate whether acidic sphingomyelinase (ASMase), a known regulator of death receptor and stress-induced hepatocyte apoptosis, plays a role in liver fibrogenesis. We show that selective stimulation of ASMase (up to sixfold), but not neutral sphingomyelinase, occurs during the transdifferentiation/activation of primary mouse HSCs into myofibroblast-like cells, coinciding with cathepsin B (CtsB) and D (CtsD) processing. ASMase inhibition or genetic down-regulation by small interfering RNA blunted CtsB/D processing, preventing the activation and proliferation of mouse and human HSCs (LX2 cells). In accordance, HSCs from heterozygous ASMase mice exhibited decreased CtsB/D processing, as well as lower levels of alpha-smooth muscle actin expression and proliferation. Moreover, pharmacological CtsB inhibition reproduced the antagonism of ASMase in preventing the fibrogenic properties of HSCs, without affecting ASMase activity. Interestingly, liver fibrosis induced by bile duct ligation or carbon tetrachloride administration was reduced in heterozygous ASMase mice compared with that in wild-type animals, regardless of their sensitivity to liver injury in either model. To provide further evidence for the ASMase-CtsB pathway in hepatic fibrosis, liver samples from patients with nonalcoholic steatohepatitis were studied. CtsB and ASMase mRNA levels increased eight- and threefold, respectively, in patients compared with healthy controls. These findings illustrate a novel role of ASMase in HSC biology and liver fibrogenesis by regulating its downstream effectors CtsB/D.

Cathepsins B and D drive hepatic stellate cell proliferation and promote their fibrogenic potential.

Cathepsins have been best characterized in tumorigenesis and cell death and implicated in liver fibrosis; however, whether cathepsins directly regulate hepatic stellate cell (HSC) activation and proliferation, hence modulating their fibrogenic potential, is largely unknown. Here, we show that expression of cathepsin B (CtsB) and cathepsin D (CtsD) is negligible in quiescent HSCs but parallels the increase of α‐smooth muscle actin and transforming growth factor‐β during in vitro mouse HSC activation. Both cathepsins are necessary for HSC transdifferentiation into myofibroblasts, because their silencing or inhibition decreased HSC proliferation and the expression of phenotypic markers of HSC activation, with similar results observed with the human HSC cell line LX2. CtsB inhibition blunted AKT phosphorylation in activated HSCs in response to platelet‐derived growth factor. Moreover, during in vivo liver fibrogenesis caused by CCl4 administration, CtsB expression increased in HSCs but not in hepatocytes, and its inactivation mitigated CCl4‐induced inflammation, HSC activation, and collagen deposition. Conclusion: These findings support a critical role for cathepsins in HSC activation, suggesting that the antagonism of cathepsins in HSCs may be of relevance for the treatment of liver fibrosis. (HEPATOLOGY 2009.)

ASMase is required for chronic alcohol induced hepatic endoplasmic reticulum stress and mitochondrial cholesterol loading

BACKGROUND & AIMS The pathogenesis of alcohol-induced liver disease (ALD) is poorly understood. Here, we examined the role of acid sphingomyelinase (ASMase) in alcohol induced hepatic endoplasmic reticulum (ER) stress, a key mechanism of ALD. METHODS We examined ER stress, lipogenesis, hyperhomocysteinemia, mitochondrial cholesterol (mChol) trafficking and susceptibility to LPS and concanavalin-A in ASMase(-)(/-) mice fed alcohol. RESULTS Alcohol feeding increased SREBP-1c, DGAT-2, and FAS mRNA in ASMase(+/+) but not in ASMase(-/-) mice. Compared to ASMase(+/+) mice, ASMase(-/-) mice exhibited decreased expression of ER stress markers induced by alcohol, but the level of tunicamycin-mediated upregulation of ER stress markers and steatosis was similar in both types of mice. The increase in homocysteine levels induced by alcohol feeding was comparable in both ASMase(+/+) and ASMase(-/-) mice. Exogenous ASMase, but not neutral SMase, induced ER stress by perturbing ER Ca(2+) homeostasis. Moreover, alcohol-induced mChol loading and StARD1 overexpression were blunted in ASMase(-/-) mice. Tunicamycin upregulated StARD1 expression and this outcome was abrogated by tauroursodeoxycholic acid. Alcohol-induced liver injury and sensitization to LPS and concanavalin-A were prevented in ASMase(-/-) mice. These effects were reproduced in alcohol-fed TNFR1/R2(-/-) mice. Moreover, ASMase does not impair hepatic regeneration following partial hepatectomy. Of relevance, liver samples from patients with alcoholic hepatitis exhibited increased expression of ASMase, StARD1, and ER stress markers. CONCLUSIONS Our data indicate that ASMase is critical for alcohol-induced ER stress, and provide a rationale for further clinical investigation in ALD.

ASMase regulates autophagy and lysosomal membrane permeabilization and its inhibition prevents early stage non-alcoholic steatohepatitis

BACKGROUND & AIMS Acid sphingomyelinase (ASMase) is activated in non-alcoholic steatohepatitis (NASH). However, the contribution of ASMase to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined the role of ASMase in high fat diet (HFD)-induced NASH. METHODS Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase(-/-) mice fed a HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed a HFD. RESULTS ASMase deficiency determined resistance to hepatic steatosis mediated by a HFD or methionine-choline deficient diet. ASMase(-/-) mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress, indicating selectivity in the resistance of ASMase(-/-) mice to ER stress and steatosis. Autophagic flux, determined in the presence of rapamycin and/or chloroquine, was lower in primary mouse hepatocytes (PMH) from ASMase(-/-) mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldin A caused ER stress in PMH from ASMase(+/+) mice but not in ASMase(-/-) mice. ASMase(-/-) PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD-induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH. CONCLUSIONS These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH.

Cathepsin B Overexpression Due to Acid Sphingomyelinase Ablation Promotes Liver Fibrosis in Niemann-Pick Disease

Background: The mechanism of liver fibrosis in Niemann-Pick disease (NPD) is unknown. Results: The loss of ASMase stimulates cathepsin B (CtsB) activation promoting liver fibrosis. Conclusion: CtsB contributes to the hepatic phenotype of NPD. Significance: CtsB may be a novel therapeutic target to treat liver disease in NPD. Niemann-Pick disease (NPD) is a lysosomal storage disease caused by the loss of acid sphingomyelinase (ASMase) that features neurodegeneration and liver disease. Because ASMase-knock-out mice models NPD and our previous findings revealed that ASMase activates cathepsins B/D (CtsB/D), our aim was to investigate the expression and processing of CtsB/D in hepatic stellate cells (HSCs) from ASMase-null mice and their role in liver fibrosis. Surprisingly, HSCs from ASMase-knock-out mice exhibit increased basal level and activity of CtsB as well as its in vitro processing in culture, paralleling the enhanced expression of fibrogenic markers α-smooth muscle actin (α-SMA), TGF-β, and pro-collagen-α1(I) (Col1A1). Moreover, pharmacological inhibition of CtsB blunted the expression of α-SMA and Col1A1 and proliferation of HSCs from ASMase-knock-out mice. Consistent with the enhanced activation of CtsB in HSCs from ASMase-null mice, the in vivo liver fibrosis induced by chronic treatment with CCl4 increased in ASMase-null compared with wild-type mice, an effect that was reduced upon CtsB inhibition. In addition to liver, the enhanced proteolytic processing of CtsB was also observed in brain and lung of ASMase-knock-out mice, suggesting that the overexpression of CtsB may underlie the phenotype of NPD. Thus, these findings reveal a functional relationship between ASMase and CtsB and that the ablation of ASMase leads to the enhanced processing and activation of CtsB. Therefore, targeting CtsB may be of relevance in the treatment of liver fibrosis in patients with NPD.

Glucose dependence of glycogen synthase activity regulation by GSK3 and MEK/ERK inhibitors and angiotensin-(1-7) action on these pathways in cultured human myotubes.

Glycogen synthase (GS) is activated by glucose/glycogen depletion in skeletal muscle cells, but the contributing signaling pathways, including the chief GS regulator GSK3, have not been fully defined. The MEK/ERK pathway is known to regulate GSK3 and respond to glucose. The aim of this study was to elucidate the GSK3 and MEK/ERK pathway contribution to GS activation by glucose deprivation in cultured human myotubes. Moreover, we tested the glucose-dependence of GSK3 and MEK/ERK effects on GS and angiotensin (1-7) actions on these pathways. We show that glucose deprivation activated GS, but did not change phospho-GS (Ser640/1), GSK3β activity or activity-activating phosphorylation of ERK1/2. We then treated glucose-replete and -depleted cells with SB415286, U0126, LY294 and rapamycin to inhibit GSK3, MEK1/2, PI3K and mTOR, respectively. SB415286 activated GS and decreased the relative phospho-GS (Ser640/1) level, more in glucose-depleted than -replete cells. U0126 activated GS and reduced the phospho-GS (Ser640/1) content significantly in glucose-depleted cells, while GSK3β activity tended to increase. LY294 inactivated GS in glucose-depleted cells only, without affecting relative phospho-GS (Ser640/1) level. Rapamycin had no effect on GS activation. Angiotensin-(1-7) raised phospho-ERK1/2 but not phospho-GSK3β (Ser9) content, while it inactivated GS and increased GS phosphorylation on Ser640/1, in glucose-replete cells. In glucose-depleted cells, angiotensin-(1-7) effects on ERK1/2 and GS were reverted, while relative phospho-GSK3β (Ser9) content decreased. In conclusion, activation of GS by glucose deprivation is not due to GS Ser640/1 dephosphorylation, GSK3β or ERK1/2 regulation in cultured myotubes. However, glucose depletion enhances GS activation/Ser640/1 dephosphorylation due to both GSK3 and MEK/ERK inhibition. Angiotensin-(1-7) inactivates GS in glucose-replete cells in association with ERK1/2 activation, not with GSK3 regulation, and glucose deprivation reverts both hormone effects. Thus, the ERK1/2 pathway negatively regulates GS activity in myotubes, without involving GSK3 regulation, and as a function of the presence of glucose.

Paracrine cellular senescence exacerbates biliary injury and impairs regeneration

Cellular senescence is a mechanism that provides an irreversible barrier to cell cycle progression to prevent undesired proliferation. However, under pathological circumstances, senescence can adversely affect organ function, viability and regeneration. We have developed a mouse model of biliary senescence, based on the conditional deletion of Mdm2 in bile ducts under the control of the Krt19 promoter, that exhibits features of biliary disease. Here we report that senescent cholangiocytes induce profound alterations in the cellular and signalling microenvironment, with recruitment of myofibroblasts and macrophages causing collagen deposition, TGFβ production and induction of senescence in surrounding cholangiocytes and hepatocytes. Finally, we study how inhibition of TGFβ-signalling disrupts the transmission of senescence and restores liver function. We identify cellular senescence as a detrimental mechanism in the development of biliary injury. Our results identify TGFβ as a potential therapeutic target to limit senescence-dependent aggravation in human cholangiopathies.Senescence has been suggested as causing biliary cholangiopathies but how this is regulated is unclear. Here, the authors generate a mouse model of biliary senescence by deleting Mdm2 in bile ducts and show that inhibiting TGFβ limits senescence-dependent aggravation of cholangiopathies.

1059 CRITICAL ROLE OF TNF-RECEPTOR 1 BUT NOT 2 IN HEPATIC STELLATE CELL PROLIFERATION, EXTRACELLULAR MATRLX REMODELING AND LIVER FIBROGENESIS

Trabajo presentado en el 46th Annual Meeting of the European Association for the Study of the Liver (EASL), celebrado en Berlin, Alemania, del 30 de marzo al 3 de abril de 2011

Hepatic stellate cell proliferation. A potential role of protein kinase R: Reply

We read with great interest the article by Martinot-Peignoux et al. In this report from France, undetectable serum hepatitis C virus (HCV) RNA at 12 weeks (Wþ12) (409 patients) post-treatment follow-up was as relevant as undetectable serum HCV RNA at 24 weeks (Wþ24) (sustained virological response [SVR]; 408 patients) after the end of treatment. Current standard therapy is based on a combination of pegylated interferon (PEG-IFN) and ribavirin, but it leads to only 50% SVR in patients with HCV genotype 1 and high viral loads. IFN reduced the risk for HCC, especially among patients with SVR. Then, we need to accurately judge whether the patient is SVR or non-SVR, applying the present standard for the judgment of SVR with the undetectability of serum HCV RNA at post-treatment Wþ24. We investigated 102 patients with chronic hepatitis C genotype 1 treated with PEG-IFN-alfa 2a plus ribavirin for 48 weeks. Some of these patients had already been included in previous reports. Serum HCV RNA was measured using the COBAS TaqMan HCV test with a detection limit of 1.2 logIU/mL. At the Wþ24 posttreatment follow-up, 40 (39.2%) patients had SVR, and 31 (48.4%) and 9 (23.6%) were treatment naı̈ve and previously treated patients, respectively. At Wþ12, serum HCV RNA was undetectable in 42 patients, and 40 patients were SVR (PPV, 95.2%). We found two relapsers at Wþ24 (undetectable at Wþ12). In the case of using direct-acting antivirals, earlier knowledge of treatment outcome would be useful for retreatment for the same patient. Taken together, our findings show that Wþ12 undetectable serum HCV RNA is not suitable for predicting persistent virological response. Further understanding of the mechanism of relapse could be useful in reducing the posttreatment follow-up period from the current standard of 24 weeks.