Arginase 1 expression is increased during hepatic stellate cell activation and facilitates collagen synthesis

Activation of hepatic stellate cells (HSC) is a key event in the initiation of liver fibrosis. Activated HSCs proliferate and secrete excessive amounts of extracellular matrix (ECM), disturbing liver architecture and function, leading to fibrosis and eventually cirrhosis. Collagen is the most abundant constituent of ECM and proline is the most abundant amino acid of collagen. Arginine is the precursor in the biosynthetic pathway of proline. Arginine is the exclusive substrate of both nitric oxide synthase (NOS) and arginase. NOS is an M1 (proinflammatory) marker of macrophage polarization whereas arginase-1 (Arg1) is an M2 (profibrogenic) marker of macrophage polarization. Differential expression of NOS and Arg1 has not been studied in HSCs yet. To identify the expression profile of arginine catabolic enzymes during HSC activation and to investigate their role in HSC activation, primary rat HSCs were cultured-activated for 7 days and expression of iNOS and Arg1 were investigated. Nor-NOHA was used as a specific and reversible arginase inhibitor. During HSC activation, iNOS expression decreased whereas Arg1 expression increased. Inhibition of Arg1 in activated HSCs efficiently inhibited collagen production but not cell proliferation. HSC activation is accompanied by a switch of arginine catabolism from iNOS to Arg1. Inhibition of Arg1 decreases collagen synthesis. Therefore, we conclude that Arg1 can be a therapeutic target for the inhibition of liver fibrogenesis.</p

Hydroxyurea attenuates hepatic stellate cell proliferation in vitro and liver fibrogenesis in vivo

Liver fibrosis results from excessive proliferation of, and collagen production by hepatic stellate cells (HSCs) that is caused by chronic liver injury. No drugs are available to cure liver fibrosis. Hydroxyurea is an anti-proliferative drug that is used in benign and malignant disorders. Here, we studied the effect of hydroxyurea on primary HSCs and its anti-fibrotic effect in the CCl 4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl 4 or vehicle was administered to C57BL/6/J mice for 4 weeks, with or without hydroxyurea (100 mg/kg/day) co-treatment. We used real-time cell proliferation analysis, Oil Red O (lipid droplet) staining, immunohistochemistry, Acridine Orange staining (apoptosis), Sytox green staining (necrosis), RT-qPCR, ELISA, and Western Blotting for analysis. Hydroxyurea dose-dependently suppressed lipid droplet-loss and mRNA levels of Col1α1 and Acta2 in transdifferentiating HSCs. In fully-activated HSCs, hydroxyurea dose-dependently attenuated PCNA protein levels and BrdU incorporation, but did not reverse Col1α1 and Acta2 mRNA expression. Hydroxyurea did not induce apoptosis or necrosis in HSCs or hepatocytes. Hydroxyurea suppressed accumulation of desmin-positive HSCs and hepatic collagen deposition after CCl 4 treatment. CCl 4 -induced regenerative hepatocyte proliferation, Col1α1 and Acta2 mRNA expression and α-SMA protein levels were not affected. This study demonstrates that hydroxyurea inhibits HSC proliferation in vitro and attenuates early development of liver fibrosis in vivo, while preserving hepatocyte regeneration after toxic insults by CCl 4. Thus, hydroxyurea may have therapeutic value against liver fibrosis. </p

Effect of COPD treatments on MRP1-mediated transport in bronchial epithelial cells

Margaretha van der Deen1, Sandra Homan1, Hetty Timmer-Bosscha1, Rik J Scheper2, Wim Timens3, Dirkje S Postma4, Elisabeth G de Vries1Departments of 1Medical Oncology, 3Pathology, 4Pulmonary Diseases, University Medical Center Groningen and University of Groningen, The Netherlands; 2Department of Pathology, VU University Medical Center, Amsterdam, The NetherlandsBackground: Smoking is the principle risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is known to protect against toxic compounds and oxidative stress, and might play a role in protection against smoke-induced disease progression. We questioned whether MRP1-mediated transport is influenced by pulmonary drugs that are commonly prescribed in COPD.Methods: The immortalized human bronchial epithelial cell line 16HBE14o- was used to analyze direct in vitro effects of budesonide, formoterol, ipratropium bromide and N-acetylcysteine (NAC) on MRP1-mediated transport. Carboxyfluorescein (CF) was used as a model MRP1 substrate and was measured with functional flow cytometry.Results: Formoterol had a minor effect, whereas budesonide concentration-dependently decreased CF transport by MRP1. Remarkably, addition of formoterol to the highest concentration of budesonide increased CF transport. Ipratropium bromide inhibited CF transport at low concentrations and tended to increase CF transport at higher levels. NAC increased CF transport by MRP1 in a concentration-dependent manner.Conclusions: Our data suggest that, besides their positive effects on respiratory symptoms, budesonide, formoterol, ipratropium bromide, and NAC modulate MRP1 activity in bronchial epithelial cells. Further studies are required to assess whether stimulation of MRP1 activity is beneficial for long-term treatment of COPD.Keywords: bronchus epithelium, COPD, drugs, MRP1, multidrug resistance, oxidative stres

Hepatic stellate cells induce an inflammatory phenotype in Kupffer cells via the release of extracellular vesicles

Liver fibrosis is the response of the liver to chronic liver inflammation. The communication between the resident liver macrophages (Kupffer cells [KCs]) and hepatic stellate cells (HSCs) has been mainly viewed as one-directional: from KCs to HSCs with KCs promoting fibrogenesis. However, recent studies indicated that HSCs may function as a hub of intercellular communications. Therefore, the aim of the present study was to investigate the role of HSCs on the inflammatory phenotype of KCs. Primary rat HSCs and KCs were isolated from male Wistar rats. HSCs-derived conditioned medium (CM) was harvested from different time intervals (Day 0−2: CM-D2 and Day 5−7: CM-D7) during the activation of HSCs. Extracellular vesicles (EVs) were isolated from CM by ultracentrifugation and evaluated by nanoparticle tracking analysis and western blot analysis. M1 and M2 markers of inflammation were measured by quantitative PCR and macrophage function by assessing phagocytic capacity. CM-D2 significantly induced the inflammatory phenotype in KCs, but not CM-D7. Neither CM-D2 nor CM-D7 affected the phagocytosis of KCs. Importantly, the proinflammatory effect of HSCs-derived CM is mediated via EVs released from HSCs since EVs isolated from CM mimicked the effect of CM, whereas EV-depleted CM lost its ability to induce a proinflammatory phenotype in KCs. In addition, when the activation of HSCs was inhibited, HSCs produced less EVs. Furthermore, the proinflammatory effects of CM and EVs are related to activating Toll-like receptor 4 (TLR4) in KCs. In conclusion, HSCs at an early stage of activation induce a proinflammatory phenotype in KCs via the release of EVs. This effect is absent in CM derived from HSCs at a later stage of activation and is dependent on the activation of TLR4 signaling pathway.</p

a-1 Adrenergic receptor antagonist doxazosin reverses hepatic stellate cells activation via induction of senescence

BACKGROUND: Activated hepatic stellate cells (aHSCs) are the main effector cells during liver fibrogenesis. α-1 adrenergic antagonist doxazosin (DX) was shown to be anti-fibrotic in an in vivo model of liver fibrosis (LF), but the mechanism remains to be elucidated. Recent studies suggest that reversion of LF can be achieved by inducing cellular senescence characterized by irreversible cell-cycle arrest and acquisition of the senescence-associated secretory phenotype (SASP). AIM: To elucidate the mechanism of the anti-fibrotic effect of DX and determine whether it induces senescence. METHODS: Primary culture-activated rat HSCs were used. mRNA and protein expression were measured by qPCR and Western blot, respectively. Cell proliferation was assessed by BrdU incorporation and xCelligence analysis. TGF-β was used for maximal HSC activation. Norepinephrine (NE), PMA and m-3M3FBS were used to activate alpha-1 adrenergic signaling. RESULTS: Expression of Col1α1 was significantly decreased by DX (10 µmol/L) at mRNA (-30 %) and protein level (-50 %) in TGF-β treated aHSCs. DX significantly reduced aHSCs proliferation and increased expression of senescence and SASP markers. PMA and m-3M3FBS reversed the effect of DX on senescence markers. CONCLUSION: Doxazosin reverses the fibrogenic phenotype of aHSCs and induces the senescence phenotype

Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death

Background: In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated in this activation process. Therefore, activated hepatic stellate cells need to harbor highly effective anti-oxidants to protect against the toxic effects of ROS.Aim: To investigate the protective mechanisms of activated HSCs against ROS-induced toxicity.Methods: Culture-activated rat HSCs were exposed to hydrogen peroxide. Necrosis and apoptosis were determined by Sytox Green or acridine orange staining, respectively. The hydrogen peroxide detoxifying enzymes catalase and glutathione-pefoxidase (GPx) were inhibited using 3-amino-1,2,4-triazole and mercaptosuccinic acid, respectively. The anti-oxidant glutathione was depleted by L-buthionine-sulfoximine and repleted with the GSH-analogue GSH-monoethylester (GSH-MEE).Results: Upon activation, HSCs increase their cellular glutathione content and GPx expression, while MnSOD (both at mRNA and protein level) and catalase (at the protein level, but not at the mRNA level) decreased. Hydrogen peroxide did not induce cell death in activated HSCs. Glutathione depletion increased the sensitivity of HSCs to hydrogen peroxide, resulting in 35% and 75% necrotic cells at 0.2 and 1 mmol/L hydrogen peroxide, respectively. The sensitizing effect was abolished by GSH-MEE. Inhibition of catalase or GPx significantly increased hydrogen peroxide-induced apoptosis, which was not reversed by GSH-MEE.Conclusion: Activated HSCs have increased ROS-detoxifying capacity compared to quiescent HSCs. Glutathione levels increase during HSC activation and protect against ROS-induced necrosis, whereas hydrogen peroxide-detoxifying enzymes protect against apoptotic cell death. (C) 2013 Elsevier B.V. All rights reserved.</p

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention