Therapeutic Administration of the Direct Thrombin Inhibitor Argatroban Reduces Hepatic Inflammation in Mice with Established Fatty Liver Disease
2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2012.06.011
ISSN1525-2191
AutoresKaren M. Kassel, Bradley P. Sullivan, Wei Cui, Bryan L. Copple, James P. Luyendyk,
Tópico(s)Fatty Acid Research and Health
ResumoThrombin generation is increased in patients with nonalcoholic fatty liver disease (NAFLD) and in mouse models of diet-induced obesity. Deficiency in the thrombin receptor protease activated receptor-1 reduces hepatic inflammation and steatosis in mice fed a Western diet. However, it is currently unclear whether thrombin inhibitors can modify the pathogenesis of established NAFLD. We tested the hypothesis that thrombin inhibition could reverse hepatic steatosis and inflammation in mice with established diet-induced NAFLD. Low-density lipoprotein receptor–deficient LDLr−/− mice were fed a control diet or a Western diet for 19 weeks. Mice were given the direct thrombin inhibitor argatroban ∼15 mg/kg/day or its vehicle via a miniosmotic pump for the final 4 weeks of the study. Argatroban administration significantly reduced hepatic proinflammatory cytokine expression and reduced macrophage and neutrophil accumulation in livers of mice fed a Western diet. Argatroban did not significantly impact hepatic steatosis, as indicated by histopathology, Oil Red O staining, and hepatic triglyceride levels. Argatroban reduced serum triglyceride and cholesterol levels in mice fed a Western diet. Argatroban reduced both α-smooth muscle actin expression and Type 1 collagen mRNA levels in livers of mice fed a Western diet, indicating reduced activation of hepatic stellate cells. This study indicates that therapeutic intervention with a thrombin inhibitor attenuates hepatic inflammation and several profibrogenic changes in mice fed a Western diet. Thrombin generation is increased in patients with nonalcoholic fatty liver disease (NAFLD) and in mouse models of diet-induced obesity. Deficiency in the thrombin receptor protease activated receptor-1 reduces hepatic inflammation and steatosis in mice fed a Western diet. However, it is currently unclear whether thrombin inhibitors can modify the pathogenesis of established NAFLD. We tested the hypothesis that thrombin inhibition could reverse hepatic steatosis and inflammation in mice with established diet-induced NAFLD. Low-density lipoprotein receptor–deficient LDLr−/− mice were fed a control diet or a Western diet for 19 weeks. Mice were given the direct thrombin inhibitor argatroban ∼15 mg/kg/day or its vehicle via a miniosmotic pump for the final 4 weeks of the study. Argatroban administration significantly reduced hepatic proinflammatory cytokine expression and reduced macrophage and neutrophil accumulation in livers of mice fed a Western diet. Argatroban did not significantly impact hepatic steatosis, as indicated by histopathology, Oil Red O staining, and hepatic triglyceride levels. Argatroban reduced serum triglyceride and cholesterol levels in mice fed a Western diet. Argatroban reduced both α-smooth muscle actin expression and Type 1 collagen mRNA levels in livers of mice fed a Western diet, indicating reduced activation of hepatic stellate cells. This study indicates that therapeutic intervention with a thrombin inhibitor attenuates hepatic inflammation and several profibrogenic changes in mice fed a Western diet. More than 70% of patients with abdominal obesity develop concurrent nonalcoholic fatty liver disease (NAFLD).1Bellentani S. Saccoccio G. Masutti F. Croce L.S. Brandi G. Sasso F. Cristanini G. Tiribelli C. Prevalence of and risk factors for hepatic steatosis in Northern Italy.Ann Intern Med. 2000; 132: 112-117Crossref PubMed Scopus (1078) Google Scholar NAFLD, the hepatic manifestation of metabolic syndrome, is characterized by excess accumulation of lipids in the liver (ie, hepatic steatosis)2Tiniakos D.G. Vos M.B. Brunt E.M. Nonalcoholic fatty liver disease: pathology and pathogenesis.Annu Rev Pathol. 2010; 5: 145-171Crossref PubMed Scopus (654) Google Scholar, 3Kleiner D.E. Brunt E.M. Van Natta M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. Yeh M. McCullough A.J. 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CRP and the risk of atherosclerotic events.Semin Immunopathol. 2009; 31: 79-94Crossref PubMed Scopus (95) Google Scholar are primarily dictated by the proinflammatory environment in the liver. Indeed, hs-CRP levels are independently associated with hepatic steatosis in patients with metabolic syndrome.8Ndumele C.E. Nasir K. Conceicao R.D. Carvalho J.A. Blumenthal R.S. Santos R.D. Hepatic steatosis obesity, and the metabolic syndrome are independently and additively associated with increased systemic inflammation.Arterioscler Thromb Vasc Biol. 2011; 31: 1927-1932Crossref PubMed Scopus (122) Google Scholar These studies indicate that increased hepatic inflammation is a focal point of multiple diseases stemming from the metabolic syndrome. Of importance, the molecular triggers of hepatic inflammation in metabolic diseases such as obesity are not completely understood. To this end, understanding the cellular and molecular pathways coordinating hepatic inflammation in metabolic disease could lead to the development of clinical therapies that target inflammation as an underlying cause of multiple interrelated diseases. Because the liver is the primary site of coagulation factor synthesis, liver diseases are often accompanied by a rebalancing of the hemostatic profile.19Lisman T. Porte R.J. Rebalanced hemostasis in patients with liver disease: evidence and clinical consequences.Blood. 2010; 116: 878-885Crossref PubMed Scopus (445) Google Scholar Indeed, abdominal obesity, metabolic syndrome, and NAFLD are each associated with activation of the blood coagulation cascade, including increased generation of the serine protease thrombin.20Fritsch P. Kleber M. Rosenkranz A. Fritsch M. Muntean W. Mangge H. Reinehr T. 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Rockwell C.E. Sullivan B.P. Wang R. Tawfik O. Li G. Guo G.L. Mackman N. Luyendyk J.P. Protease-activated receptor 1 and hematopoietic cell tissue factor are required for hepatic steatosis in mice fed a Western Diet.Am J Pathol. 2011; 179: 2278-2289Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Moreover, we found previously that mice lacking a thrombin receptor, protease activated receptor-1 (PAR-1), did not develop hepatic steatosis when fed a Western diet.24Kassel K.M. Owens III, A.P. Rockwell C.E. Sullivan B.P. Wang R. Tawfik O. Li G. Guo G.L. Mackman N. Luyendyk J.P. Protease-activated receptor 1 and hematopoietic cell tissue factor are required for hepatic steatosis in mice fed a Western Diet.Am J Pathol. 2011; 179: 2278-2289Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Although compelling, these genetic approaches do not directly address the question of whether intervention with pharmacological agents, perhaps anticoagulants, can reduce established liver disease. Indeed, it is currently unclear whether pharmacological inhibition of thrombin alters the course of established diet-induced fatty liver disease in mice. To this end, we tested the hypothesis that pharmacological inhibition of thrombin could therapeutically reverse diet-induced hepatic inflammation and steatosis in hypercholesterolemic low density lipoprotein receptor–deficient (LDLr−/−) mice. Six-week-old male LDLr−/− mice on a C57Bl/6 background purchased from the Jackson Laboratory (Bar Harbor, ME) were fed a control diet (AIN-93M, 10% kcal from fat; Dyets, Bethlehem, PA) or a Western diet (Diet #100244, 40% kcal from milk fat; Dyets) ad libitum for 15 weeks before implantation of a subcutaneous miniosmotic pump (see below). Mice fed each diet were then stratified to receive either vehicle or argatroban such that the body weights of each group of mice at the time of pump implantation were similar. Average food intake was measured weekly for each cage of mice, and mice were weighed weekly. All studies were approved by the Animal Care and Use Committee of the University of Kansas Medical Center and complied with National Institutes of Health guidelines. Alzet miniosmotic pumps (Model 2004, flow rate 0.25 μL/hour; Alzet, Cupertino, CA) were filled with vehicle or argatroban (AKScientific, Union City, CA) solution. Previous studies have demonstrated effective administration of argatroban to rodents via a micro-osmotic pump.27Kitaoka T. Hua Y. Xi G. Hoff J.T. Keep R.F. Delayed argatroban treatment reduces edema in a rat model of intracerebral hemorrhage.Stroke. 2002; 33: 3012-3018Crossref PubMed Scopus (119) Google Scholar, 28Mihara M. Aihara K. Ikeda Y. Yoshida S. Kinouchi M. Kurahashi K. Fujinaka Y. Akaike M. Matsumoto T. Inhibition of thrombin action ameliorates insulin resistance in type 2 diabetic db/db mice.Endocrinology. 2010; 151: 513-519Crossref PubMed Scopus (27) Google Scholar The argatroban vehicle consisted of 10% glacial acetic acid, 2 mmol/L sodium acetate, 20% polyethylene glycol 400, and 10% propylene glycol in sterile water for injection. This solution is stated to stabilize argatroban for at least 4 weeks at 37°C.29Owoo G, Burgos RA, inventors; Bayer Int, assignee. 2008 Feb 29. Argatroban formulations and methods for making and using same. United States patent US 7,915,290 B2Google Scholar Argatroban was dissolved in the vehicle solution at 100 mg/mL, yielding a daily dose of approximately 15 mg/kg/day, a dose similar to previous studies using argatroban administered to rodents via a micro-osmotic pump.27Kitaoka T. Hua Y. Xi G. Hoff J.T. Keep R.F. Delayed argatroban treatment reduces edema in a rat model of intracerebral hemorrhage.Stroke. 2002; 33: 3012-3018Crossref PubMed Scopus (119) Google Scholar Pumps were filled in a sterile environment according to the manufacturer's protocol. Mice were anesthetized with 3% isoflurane and given an intraperitoneal injection of Buprenex (buprenorphine HCl, 1 mg/kg; Reckitt Benckiser Pharmaceuticals, Richmond, VA) before surgeries. Pumps were implanted subcutaneously slightly posterior to the scapulae, and the incision was closed with Syneture 4–0 chromic gut sutures (Roboz, Gaithersburg, MD). Sutures and incision sites were monitored daily. Mice were treated with topical zinc oxide for skin irritation as required and recommended by a clinical veterinarian. Mice were allowed ad libitum access to either control diet or Western diet (as specified above) for 28 days after pump implantation. Mice were fasted overnight before sample collection. Under isoflurane anesthesia, blood was collected from the caudal vena cava into sodium citrate (final concentration, 0.38%) for the collection of plasma and into an empty syringe for the collection of serum. Sections of liver from the left lateral lobe were fixed in 10% neutral-buffered formalin for 48 hours and embedded in paraffin. The right medial lobe was affixed to a cork with optimal cutting temperature compound (Thermo Fisher Scientific, Waltham, MA) and immersed for approximately 3 minutes in liquid nitrogen–chilled isopentane. The remaining liver was snap frozen in liquid nitrogen. Paraffin-embedded livers were sectioned at 5 μm and stained with hematoxylin and eosin (H&E). Livers were scored by a board-certified pathologist (W.C.) for percent steatosis and the presence of inflammatory foci. The total number of foci of portal inflammation and lobular inflammation was counted for the entire section under ×200 magnification. The raw score of inflammatory foci per ×200 field was calculated by dividing the total number of inflammatory foci by the total number of fields. Formalin-fixed sections were also stained for collagen using a Chromaview Gomori Trichrome staining kit (Thermo Fisher Scientific) as described by the manufacturer's protocol. Oil Red O staining was performed as previously described.30Tanaka Y. Aleksunes L.M. Yeager R.L. Gyamfi M.A. Esterly N. Guo G.L. Klaassen C.D. NF-E2-related factor 2 inhibits lipid accumulation and oxidative stress in mice fed a high-fat diet.J Pharmacol Exp Ther. 2008; 325: 655-664Crossref PubMed Scopus (202) Google Scholar Quantification of collagen staining (blue) and Oil Red O staining was performed using Metamorph software (Molecular Devices, Sunnyvale, CA) and ImageJ version 1.45 (NIH, Bethesda, MD). Plasma thrombin-antithrombin levels were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Siemens Healthcare Diagnostics, Tarrytown, NY). Lipids were extracted from 100 mg of snap-frozen liver as described,31Luyendyk J.P. Sullivan B.P. Guo G.L. Wang R. Tissue factor-deficiency and protease activated receptor-1-deficiency reduce inflammation elicited by diet-induced steatohepatitis in mice.Am J Pathol. 2010; 176: 177-186Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar and hepatic and serum triglyceride and cholesterol levels were determined using commercially available reagents (Pointe Scientific, Canton, MI). Thrombin time was determined by clotting 50 μL of mouse plasma with 50 μL of Dade thrombin reagent (6.25 U/mL; Siemens Healthcare Diagnostics) using a Start4 coagulation analyzer (Diagnostica Stago, Parsippany, NJ). Total RNA was isolated from approximately 100 mg of snap-frozen liver using TRI Reagent (Molecular Research Center, Cincinnati, OH) per the manufacturer's protocol. One microgram of RNA was used for the synthesis of cDNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) and C1000 thermal cycler (Bio-Rad Laboratories, Hercules, CA). Levels of MCP-1, CD68, F4/80, intercellular adhesion molecule-1 (ICAM-1), macrophage inflammatory protein-2 (MIP-2), type 1 collagen (Col1a1), tissue inhibitor of metalloproteinases-1 (TIMP-1), transforming growth factor-β1 (TGFβ1), and 18S mRNA were determined using either TaqMan gene expression assays (Applied Biosystems) or PrimeTime qPCR Assays [Integrated DNA Technologies (IDT), Coralville, IA], iTaq Supermix with ROX (Bio-Rad), and a StepOnePlus thermal cycler (Applied Biosystems). Mouse MIP-2 (NM_009140) primer sequences were 5′-GAAGTCATAGCCACTCTCAAGG-3′ (forward primer), 5′-CTTCCGTTGAGGGACAGC-3′ (reverse primer), and 5′-/56-FAM/TCCTTTCCA/ZEN/GGTCAGTTAGCCTTGC/3IABkFQ/-3′ (probe). Mouse Col1a1 (NM_007742) primer sequences were 5′-CATAAAGGGTCATGGTGGCT-3′ (forward primer), 5′-TTGAGTCCGTCTTTGCCAG-3′ (reverse primer), and 5′-/56-FAM/TGGTGAACA/ZEN/AGGCCCCTCTGG/3IABkGQ/-3′ (probe). 18S (NM_003286) primer sequences were 5′-CTGTAGCCCTGTACTTCATCG-3′ (forward primer), 5′-CTACCACATATTCCTGACCATCC-3′ (reverse primer), and 5′-/56-FAM/CCTTCCTCC/ZEN/TTTTCATTGCCTGCTCT/3IABkFQ/-3′ (probe). MIP-2, Col1a1, and 18S primers and probes were purchased from IDT. All other gene expression assays were purchased from Applied Biosystems (MCP-1, Assay ID Mm00441242_m1; CD68, Assay ID Mm00839636_g1; F4/80, Assay ID Mm00802530_m1; ICAM-1, Assay ID Mm00516023_m1; TIMP-1, Assay ID Mm00441818_m1; and TGFβ1, Assay ID Mm00441724_m1). The expression of each gene was adjusted relative to 18S expression levels, and the relative expression level was determined using the comparative Ct method. Frozen livers were sectioned at 8 μm for macrophage staining. Macrophages were identified in liver using CD68 and F4/80 antibodies as previously described.31Luyendyk J.P. Sullivan B.P. Guo G.L. Wang R. Tissue factor-deficiency and protease activated receptor-1-deficiency reduce inflammation elicited by diet-induced steatohepatitis in mice.Am J Pathol. 2010; 176: 177-186Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Paraffin-embedded livers were sectioned at 5 μm and stained for neutrophils by the Michigan State University Investigative HistoPathology Laboratory using a rabbit anti-neutrophil polyclonal antibody. The total number of neutrophils and the number of neutrophil foci (defined as three or more adjacent neutrophils) per 10 randomly selected ×200 fields were determined. Paraffin-embedded livers were sectioned at 5 μm, deparaffinized, and then boiled in citrate buffer. Sections were blocked with 5% goat serum and stained overnight with rabbit anti–α-smooth muscle actin (αSMA) antibody (Abcam, Cambridge, MA). Tissues were washed with Tris-buffered saline–Tween and stained with biotinylated anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 30 minutes. ABC reagent (Vector Laboratories, Burlingame, CA) was added for 30 minutes, and tissues were then incubated with ImmPACT DAB (Vector Laboratories) for 1 minute. Sections were counterstained with Gill's hematoxylin (Ricca Chemical Company, Arlington, TX) and Scott's Bluing Reagent (Ricca Chemical Company). Quantification of αSMA staining was performed using Metamorph software (Molecular Devices). Total protein was isolated from approximately 100 mg of snap-frozen liver using PBS containing 0.1% Triton X-100 and Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Samples were rotated for 30 minutes at 4°C and then subjected to centrifugation at 12,000 × g for 15 minutes at 4°C. Protein concentrations were determined using a DC Protein Assay (Bio-Rad). The concentrations of MCP-1 in liver and plasma were determined using a commercially available ELISA kit (DuoSet ELISA; R&D Systems, Minneapolis, MN). Statistics were performed using SigmaPlot 11.0 software (Systat Software, San Jose, CA). Comparison of two groups was performed using Student's t-test. Comparison of three or more groups was performed using two-way analysis of variance followed by the Student-Newman-Keuls post hoc test for multiple comparisons. Data are expressed as mean ± SEM. The criterion for statistical significance was P < 0.05. In agreement with our previous studies,24Kassel K.M. Owens III, A.P. Rockwell C.E. Sullivan B.P. Wang R. Tawfik O. Li G. Guo G.L. Mackman N. Luyendyk J.P. Protease-activated receptor 1 and hematopoietic cell tissue factor are required for hepatic steatosis in mice fed a Western Diet.Am J Pathol. 2011; 179: 2278-2289Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar plasma thrombin-antithrombin levels, a stable biomarker of thrombin generation, significantly increased in mice fed a Western diet compared to mice fed a control diet (data not shown). To confirm thrombin inhibition by argatroban, plasma thrombin time was determined. Argatroban treatment significantly prolonged thrombin time (43.3 ± 6.2 seconds) compared to mice treated with vehicle (27.9 ± 1.2 seconds) (P < 0.05). These studies suggest that thrombin generation is increased in mice fed a Western diet and that thrombin activity was reduced in mice treated with argatroban. Mice fed a control diet gained 42.1 ± 2.7% body weight, and mice fed a Western diet gained 97.1 ± 6.7% body weight in the 15 weeks before pump implantation (P < 0.05). Following pump implantation, argatroban treatment did not affect weight gain in mice fed either diet (data not shown). In addition, argatroban treatment did not significantly affect food intake (data not shown). No overt bleeding events were noted in mice treated with argatroban. Argatroban administration significantly reduced hepatic macrophage accumulation in mice fed a Western diet (Figure 1, A–D). In agreement, hepatic expression of the mRNAs encoding CD68 and F4/80, two macrophage-selective genes, was significantly reduced by argatroban administration in mice fed a Western diet (Figure 1, E and F). MCP-1 is an inflammatory mediator that contributes to hepatic macrophage accumulation and the development of steatosis in mice fed a Western diet.11Tamura Y. Sugimoto M. Murayama T. Minami M. Nishikaze Y. Ariyasu H. Akamizu T. Kita T. Yokode M. Arai H. C-C chemokine receptor 2 inhibitor improves diet-induced development of insulin resistance and hepatic steatosis in mice.J Atheroscler Thromb. 2010; 17: 219-228Crossref PubMed Scopus (71) Google Scholar, 12Rull A. Rodriguez F. Aragones G. Marsillach J. Beltran R. Alonso-Villaverde C. Camps J. Joven J. Hepatic monocyte chemoattractant protein-1 is upregulated by dietary cholesterol and contributes to liver steatosis.Cytokine. 2009; 48: 273-279Crossref PubMed Scopus (41) Google Scholar, 32Clement S. Juge-Aubry C. Sgroi A. Conzelmann S. Pazienza V. Pittet-Cuenod B. Meier C.A. Negro F. Monocyte chemoattractant protein-1 secreted by adipose tissue induces direct lipid accumulation in hepatocytes.Hepatology. 2008; 48: 799-807Crossref PubMed Scopus (70) Google Scholar, 33Endo M. Masaki T. Seike M. Yoshimatsu H. TNF-alpha induces hepatic steatosis in mice by enhancing gene expression of sterol regulatory element binding protein-1c (SREBP-1c).Exp Biol Med (Maywood). 2007; 232: 614-621PubMed Google Scholar, 34De Taeye B.M. Novitskaya T. McGuinness O.P. Gleaves L. Medda M. Covington J.W. Vaughan D.E. Macrophage TNF-alpha contributes to insulin resistance and hepatic steatosis in diet-induced obesity.Am J Physiol Endocrinol Metab. 2007; 293: E713-E725Crossref PubMed Scopus (164) Google Scholar Compared to LDLr−/− mice fed a control diet, hepatic MCP-1 mRNA, as well as hepatic and plasma MCP-1 protein levels, was increased in LDLr−/− mice fed a Western diet (Figure 2, A–C). Administration of argatroban for 4 weeks significantly reduced MCP-1 mRNA and protein expression in mice fed a Western diet (Figure 2, A–C).Figure 2Effect of thrombin inhibition on MCP-1 induction in mice fed a Western diet. LDLr−/− mice were fed a control diet or a Western diet for 19 weeks and were treated with vehicle or argatroban (15 mg/kg/day) via a miniosmotic pump for the final 4 weeks of the study. A: Hepatic levels of MCP-1 mRNA were determined by real-time PCR. Data are expressed as mean ± SEM and as a fold change versus mice fed control diet with a vehicle pump. B: Hepatic levels and (C) plasma levels of MCP-1 protein were determined by ELISA. Data are expressed as mean ± SEM. n = 5 to 7 mice per group. *P < 0.05 versus mice fed the control diet with the same drug treatment. †P < 0.05 versus mice fed the same diet with a vehicle pump.View Large Image
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