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doi:10.1111/jgh.12720
H E PAT O L O G Y
Inconsistent hepatic antifibrotic effects with the iron chelator deferasirox Amy Sobbe,* Kim R Bridle,* Lesley Jaskowski,* C Erika de Guzman,* Nishreen Santrampurwala,* Andrew D Clouston,† Catherine M Campbell,† V Nathan Subramaniam*,‡ and Darrell H G Crawford* *School of Medicine, The University of Queensland, Gallipoli Medical Research Foundation, Greenslopes Private Hospital, †Envoi Specialist Pathologists, and ‡Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
Key words animal models, iron-chelating agents, liver fibrosis, stellate cells. Accepted for publication 17 August 2014. Correspondence Dr Kim R Bridle, School of Medicine, The University of Queensland, Lower Lobby Level, Administration Building, Greenslopes Private Hospital, Greenslopes, Qld 4120, Australia. Email: [email protected]
Abstract Background and Aim: Development of effective antifibrotic treatments that can be translated to clinical practice is an important challenge in contemporary hepatology. A recent report on β-thalassemia patients demonstrated that deferasirox treatment reversed or stabilized liver fibrosis independent of its iron-chelating properties. In this study, we investigated deferasirox in cell and animal models to better understand its potential antifibrotic effects. Methods: The LX-2 stellate cell line was treated with 5 μM or 50 μM deferasirox (Exjade, Novartis Pharmaceuticals Australia, North Ryde, NSW, Australia) for up to 120 h. Three-week-old multidrug resistance 2 null (Mdr2–/–) mice received oral deferasirox or vehicle for 4 weeks (30 mg/kg/day). Cells and liver tissue were collected for assessment of fibrosis and fibrogenic gene expression. Results: In LX-2 cells treated with 50 μM deferasirox for 12 h, α1(I)procollagen expression was decreased by 25%, with maximal reductions (10-fold) seen following 24–120 h of treatment. Similarly, α-smooth muscle actin (αSMA) expression was significantly lower. Alterations in matrix remodeling genes, specifically decreased expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2, were observed. There was no significant difference in hepatic hydroxyproline content in Mdr2–/– mice following deferasirox administration (vehicle: 395 ± 27 μg/g vs deferasirox: 421 ± 33 μg/g). Similarly, no changes in the expression of fibrogenic genes were observed. Conclusion: Despite reductions in α1(I)procollagen and αSMA expression and alterations in matrix degradation genes in LX-2 cells, deferasirox did not exhibit antifibrotic activity in Mdr2–/– mice. Given the positive outcomes seen in human trials, it may be appropriate to study deferasirox in other animal models of fibrosis and/or for a longer duration of therapy.
Introduction With the increasing prevalence of liver disease, antifibrotic therapies that can be translated to clinical practice are urgently needed. One agent that has shown beneficial effects on hepatic fibrosis in patients is deferasirox, an oral iron chelator used primarily for the management of transfusional iron overload in patients with chronic anemias (reviewed in Lindsey and Olin1 and Yang et al.2). In these patients, long-term transfusions result in accumulation of iron in the body, predominantly in the liver and heart.3 The consequences of excess hepatic iron include oxidative damage to cellular proteins and membrane lipids and, for many patients, hepatic fibrosis or cirrhosis.4 Deferasirox complexes with iron in a 2:1 (deferasirox : iron) ratio and is highly selective for Fe3+ over Fe2+.2 Chelation with 638
deferasirox reduces hepatic iron concentration, serum ferritin, non-transferrin-bound iron (NTBI)5,6 and reduces cardiac siderosis.7,8 However, Deugnier et al.9 have shown that deferasirox has antifibrotic activity independent of its iron-chelating properties. In β-thalassemia patients, treatment with deferasirox was associated with stability or improvement in liver fibrosis and a reduction in serum alanine aminotransferase (ALT) levels. In many patients, a reduction in Ishak fibrosis stage was seen without a reduction in hepatic iron concentration. Despite these favorable indications in patients, there have been limited studies investigating deferasirox as an antifibrotic agent in either cell culture or animal models of hepatic fibrosis that are not complicated by iron overload and the mechanisms responsible are largely unexplored. Multidrug resistance 2 null (Mdr2–/–) mice, a model of cholestatic liver disease, lack the cannalicular phospholipid flippase, and have significantly
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altered expression of fibrogenic genes by 4 weeks of age and fully developed fibrosis by 10 weeks of age.10 These mice provide an ideal model to study potential therapeutics for hepatic fibrosis. Similarly, the LX-2 cell (a human hepatic stellate cell [HSC] line)11 is an in vitro model suitable for studies on therapeutics for fibrosis. Thus, we investigated deferasirox therapy in LX-2 cells and in the Mdr2–/– mouse model of hepatic fibrosis to determine its potential antifibrotic activity.
Methods Cell culture. LX-2 cells11 were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Musgrave, Vic., Australia) supplemented with 10% fetal calf serum, 1% penicillin/ streptomycin, and 1% glutamax. Cells were cultured with deferasirox (Novartis Pharmaceuticals Australia, North Ryde, NSW, Australia) or vehicle (water) for 3, 6, 12, 24, 48, 72, 96, and 120 h.
Proliferation and viability. Cell proliferation was measured using a CellTiter96 Aqueous One Solution Cell Proliferation Assay (Promega, Alexandria, NSW, Australia), and cell viability was measured using a CellTiter-Blue Cell Viability Assay (Promega) as per the manufacturer’s instructions. Absorbance was measured using an Infinite F200 Plate Reader (Tecan, Port Melbourne, Vic., Australia).
Animals. Male and female Mdr2–/– mice (FVB-N background, The Jackson Laboratory, Bar Harbor, ME, USA) were used for these studies. Mice were kept under 12 h light/dark cycles and given ad libitum access to a standard mouse diet (Specialty Feeds, Glen Forrest, WA, Australia) and water. Mdr2–/– mice (four male and four female per group) were treated with vehicle (0.5% hydroxypropylcellulose) or deferasirox (30 mg/kg/day) by daily oral gavage from 3 to 7 weeks of age. This dose of deferasirox has previously been shown to have limited effects on tissue iron levels in a mouse model12 and allowed us to assess antifibrotic activity independent of iron chelation. The study was approved by the University of Queensland Animal Ethics Committee and performed according to the Australian code for the care and use of animals for scientific purposes.
Assessment of iron stores. Hepatic and splenic iron concentrations were measured as previously described.13 Serum iron, unsaturated iron-binding capacity, transferrin saturation, and total iron-binding capacity (TIBC) were measured with an Iron/TIBC Reagent Set (Pointe Scientific, Canton, MI, USA) according to the manufacturer’s instructions but scaled down to suit a 96-well plate format.
Serum biochemistry. Liver function tests were performed on serum to evaluate ALT, alkaline phosphatase (ALP) and bilirubin levels as per manufacturer’s instructions (Bioo Scientific, Austin, TX, USA).
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Determination of hydroxyproline content. The hepatic concentration of hydroxyproline (HP) was measured using the method of Jamall et al.14 with modifications.15 HP levels were calculated from a standard curve of trans-4-hydroxy-L-proline run concurrently and expressed relative to tissue weight. Histological evaluation. Paraffin-embedded liver sections were used to quantify fibrosis, inflammation, and ductular reaction after HE and Sirius red staining. Sections were reviewed independently by two experienced histopathologists blinded to laboratory data and staged and graded according to accepted criteria and as published previously.16–18 RNA isolation and quantitative real-time polymerase chain reaction analysis. Isolation of total RNA was performed using TrizolTM Reagent (Life Technologies, Mulgrave, VIC, Australia) as per the manufacturer’s instructions and as previously described.19,20 Quantitative real-time polymerase chain reaction (PCR) using Sybr Green (Life Technologies) was performed on a Viia7 Real-Time PCR System (Life Technologies). Mouse and human primer nucleotide sequences and accession numbers can be found in Table S1. Results for each sample were calculated as a percentage of the β2-microglobulin (β2-m) and basic transcription factor 3 (BTF3) mRNA concentration (mouse samples) or of β2-microglobulin (β2-m) and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA concentration (LX-2 samples). Zymography. Ten micrograms of protein extract from liver was assayed for matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) activity by gelatin zymography as previously described.16,21 The relative activities of MMP-2 and MMP-9 for each sample were visualized using a Carestream Image Station 4000 MM Pro and quantified using Carestream Molecular Imaging software (Carestream, East Melbourne, Vic., Australia). Statistical analysis. Statistical analysis was performed using IBM SPSS Statistics 21 (IBM Corporation, New York, NY, USA). For in vitro studies at each timepoint, one-way anova analysis was performed. Where this global test indicated statistical significance, least significant differences post-hoc analysis was used to determine differences between treatment groups. For in vivo studies, differences between vehicle and deferasirox treated groups were assessed using a Students’ t-test. For all analyses, a P-value < 0.05 was considered statistically significant.
Results Effect of deferasirox on LX-2 cells. LX-2 cells were treated with 0, 5, or 50 μM deferasirox for 6 or 24 h. Proliferation and viability of cells was measured using the CellTiter96 Aqueous One Solution Cell Proliferation Assay and CellTiter-Blue Cell Viability Assay. No significant differences in LX-2 cell proliferation (P = 0.087 and 0.238) or viability (P = 0.194 and 0.664) were seen at either 6 or 24 h following deferasirox treatment (Fig. 1). To determine the effect of deferasirox on cellular iron status we measured transferrin receptor 1 (TfR1) expression, and to examine markers of oxidative stress we assessed gene expression of antioxidants glutathione peroxidase 1 (GPx1), manganese superoxide
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dismutase 2 (SOD2), and thioredoxin 1 (TXN). TfR1 expression has been used previously as a surrogate marker of cellular iron levels.22 Following 3–72 h deferasirox treatment, there were no significant changes in TfR1 expression compared with vehicle alone. Deferasirox treatment at 96 and 120 h resulted in increased TfR1 expression (96 h: 1.7-fold, P = 0.01 and 2.0-fold, P < 0.001 for 5 and 50 μM deferasirox, respectively, and 120 h: 1.8-fold, P = 0.02 and 1.3-fold, P = 0.05 for 5 and 50 μM deferasirox, respectively; results not shown). Following 5 μM deferasirox treatment, GPx1 expression was increased compared with vehicle at 24 h (1.2-fold, P = 0.002) and decreased at 48 h (0.9-fold, P = 0.03); SOD2 was decreased at 72, 96, and 120 h (0.8-fold, 0.7-fold, 0.6-fold; P = 0.01, 0.001, 0.02, respectively), and TXN gene expression was reduced at 48 and 120 h (0.9-fold and 0.8-fold, P = 0.03 and 0.03, respectively). Deferasirox treatment (50 μM) decreased GPx1 expression at 6–48 h (0.85-fold, 0.7-fold, 0.8-fold, 0.6-fold, P = 0.01, 0.01, 0.003, 0.001, respectively); SOD2 expression was increased at 6 and 24 h (1.2-fold and 1.2-fold, P = 0.01, 0.001, respectively) but decreased at 72 and 96 (0.8-fold, 0.7-fold, P = 0.01, 0.001 respectively), and TXN was reduced at 12, 24, and 48 h compared with vehicle (0.8-fold, 0.8-fold, 0.6-fold; P = 0.02, 0.002, 0.001, respectively; results not shown). Expression of the fibrogenic genes procollagen α1(I), (COL1A1), α-smooth muscle actin (αSMA), transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1, TIMP-2), and matrix metalloproteinase (MMP-2) were measured in LX-2 cells by qPCR at intervals from 12–120 h. COL1A1 and αSMA expression were significantly lower in LX-2 cells treated with 50 μM deferasirox for 12–120 h compared with untreated cells with maximal fold-reduction at 48 h treatment (11.5-fold and 8.3-fold, respectively; Figure 2a and c). Expression of COL1A1 and αSMA were also lower in LX-2 cells following 50 μM deferasirox compared with cells treated with 5 μM deferasirox with maximal fold-reduction seen at 48 h treatment (10.8-fold and 5.5-fold, respectively; Figure 2a and c). LX-2 cells treated with 5 μM deferasirox had lower COL1A1 and αSMA expression than untreated cells at 72, 96, and 120 h, respectively, with maximal reduction at 120 h (1.8-fold and 2.7-fold, respectively; Figure 2a and c). In contrast, at both 48 and 72 h, TGF-β1 expression was significantly higher in LX-2 cells treated with 50 μM deferasirox than in both untreated LX-2 cells (P = 0.012 and 0.003) and in those treated with 5 μM deferasirox (P = 0.002 and 0.002, Figure 2b). However, TIMP1 mRNA was significantly reduced by 50 μM deferasirox when compared with untreated cells at 96 h (1.4-fold, P = 0.011) and when compared with both 640
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Figure 1 Proliferation and viability in LX-2 cells treated with deferasirox. No significant differences were observed in LX-2 cell proliferation (a) nor LX-2 viability (b) at either 6 or 24 h following deferasirox treatment. Data are presented as the mean ± standard error of the mean (SEM) of duplicate determinations from three independent experiments. , 0 μM; , 5 μM; , 50 μM.
untreated cells and cells treated with 5 μM deferasirox at 120 h (1.4-fold, P = 0.001 and 1.2-fold, P = 0.006, respectively, Figure 2d). Similarly, TIMP2 expression was significantly reduced by 50 μM deferasirox when compared with untreated LX-2 cells after 48–120 h, with a maximal 2.6-fold-reduction at 120 h, and when compared with cells treated with 5 μM deferasirox at 48, 72, and 120 h (maximal 1.9-fold reduction at 120 h, Figure 2e). MMP2 expression was significantly lower in cells treated with 50 μM deferasirox than in untreated cells from 24–120 h treatment (P = 0.012, P = 0.001, P = 0.001, P < 0.001, and P < 0.001, respectively). MMP2 mRNA was significantly lower following 50 μM deferasirox treatment than in those treated with 5 μM deferasiox after 48 and 120 h (P = 0.018 and P = 0.001). Cells treated with 5 μM deferasirox had significantly lower MMP2 levels than untreated cells after 72, 96, and 120 h (P = 0.009, P = 0.002 and P < 0.001, respectively; Figure 2f). Body and liver weights, serum liver enzymes, and body iron stores of Mdr2–/– mice treated with deferasirox. Mdr2–/– mice were treated with deferasirox from 3 to 7 weeks of age. No significant differences in body weight (24.8 ± 1.0 vs 24.1 ± 0.8 g, P = 0.59), liver weight (2.4 ± 0.1 vs 2.3 ± 0.1 g, P = 0.56), or the liver to body weight ratio (9.6 ± 0.4 vs 9.5 ± 0.2%, P = 0.76) in Mdr2–/– mice was observed when compared with vehicle-treated animals. Similarly, there were no significant differences in serum ALT (364 ± 45 vs 361 ± 49 IU/L, P = 0.97), ALP (46 ± 4 vs 61 ± 8 IU/L, P = 0.13), or bilirubin (2.7 ± 0.7 vs 3.5 ± 0.7 g/dL, P = 0.43) levels between vehicle and deferasirox-treated groups (Table 1). Serum iron and transferrin saturation were significantly increased in Mdr2–/– mice following deferasirox treatment (300 ± 16 vs 234 ± 16 μg/dL, P = 0.015 and 39.8 ± 0.6 vs 33.2 ± 1.5%, P = 0.006, respectively; Figure 3a and b). In contrast, deferasirox treatment did not significantly alter either hepatic or splenic iron concentration (17.2 ± 1.8 vs 15.1 ± 2.0 μmol Fe/g dry weight, P = 0.478 and 71.1 ± 3.5 vs 61.1 ± 6.2 μmol Fe/g dry weight, P = 0.203, respectively; Figure 3c and d). Expression of antioxidant genes GPx1, SOD2, and TXN were not significantly different in animals treated with deferasirox compared with those given vehicle alone (0.92-fold, P = 0.6; 0.86-fold, P = 0.05; 0.9fold, P = 0.1; respectively; results not shown). Assessment of injury and fibrosis in Mdr2–/– treated with deferasirox for 4 weeks. Hepatic HP levels were similar in vehicle and deferasirox treated Mdr2–/– mice
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Figure 2 Expression of fibrogenic genes is reduced in LX-2 cells treated with deferasirox. LX-2 cells were treated with ) 0, ( ) 5, or ( ) 50 μM deferasirox for ( 3–120 h. Real-time polymerase chain reaction (RT-PCR) was used to analyze expression of (a) procollagen α1(I) (COL1A1), (b) transforming growth factor-β (TGF-β), (c) α-smooth muscle actin (αSMA), (d) tissue inhibitors of metalloproteinase-1 (TIMP1), (e) TIMP2, and (f) matrix metalloproteinase-2 MMP2. Transcript levels were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) and basic transcription factor 3 (BTF3) expression. Data are presented as the mean ± standard error of the mean (SEM) of duplicate determinations from two or three independent experiments at each time-point. *P < 0.05 50 μM versus 0 μM deferasirox, #P < 0.05 5 μM versus 0 μM deferasirox, ~P < 0.05 50 μM versus 5 μM deferasirox.
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n = 6–7. Body and liver weights were recorded at the end of the 4-week treatment period. Liver weight was expressed as a percentage of body weight. Serum liver enzymes were measured using commercial assays according to the manufacturer’s instructions. Results are expressed as mean ± standard error of the mean (SEM). ALP, alkaline phosphatase; ALT, alanine transaminase; Mdr2–/–, multidrug resistance 2 null; NS, not significant.
(395 ± 27 vs 421 ± 33 μg/g, P = 0.57, Figure 4a). Similarly, Metavir fibrosis stage (3.2 ± 0.3 vs 3.0 ± 0, P = 0.61, Figure 4b), ductular reaction score (3.8 ± 0.2 vs 4.0 0.0, P = 0.36, Figure 4c), portal inflammation (3.2 ± 0.4 vs 3.4 ± 0.2, P = 0.58, Figure 4d), and lobular inflammation (1.8 ± 0.2 vs 2.0 ± 0.4, P = 0.71, Figure 4e) and hepatocyte ballooning (1.2 ± 0.3 vs 0.6 ± 0.2, P = 0.12, Figure 4f) were not reduced following deferasirox treat-
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ment from 3 to 7 weeks of age. Representative liver sections stained with Sirius red and HE are shown in Figure 4g–j.
Fibrogenic gene expression and matrix remodelling in livers of Mdr2–/– mice treated with deferasirox. Expression of Col1a1, Tgf-β, and plateletderived growth factor receptor-B (Pdgfrb) mRNA was not significantly altered following deferasirox treatment from 3 to 7 weeks of age (P = 0.39, 0.29, and 0.71 respectively; Figure 5a–c). In contrast, αSma was increased 1.9-fold in Mdr2–/– mice following deferasirox therapy from 3 to 7 weeks of age; however, this did not reach statistical significance (P = 0.26; Figure 5d). No significant differences in Mmp2, Timp1, or Timp2 expression were noted following deferasirox treatment (P = 0.51, 0.32, and 0.68 respectively; Figure 6a–c). Similarly, gelatinase (MMP2 and MMP9) activity was not significantly different between vehicle and deferasirox-treated mice (P = 0.48 and 0.88 respectively; Figure 6d–f).
Discussion Reversal of hepatic fibrosis has been demonstrated with a variety of therapies and in injury of various aetiologies. For example, histological improvement has been observed in patients with hepa-
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Figure 3 Deferasirox treatment does not reduce serum, hepatic, and splenic iron levels in multidrug resistance 2 null (Mdr2–/–) mice. (a) Serum iron, (b) transferrin saturation, and (c) hepatic and (d) splenic iron concentrations were measured in Mdr2–/– mice treated with vehicle or deferasirox from 3 to 7 weeks of age. Results are expressed as mean ± standard error of the mean (SEM). n = 6–7. *P < 0.05 vehicle versus deferasirox.
titis C and D who have sustained response to interferon therapy23–25 and in patients who have received long-term venesection for hemochromatosis.26 These therapies primarily target the cause of injury whereas agents that inhibit the fibrogenic process have shown little efficacy in patients. Deugnier et al.9 have demonstrated that deferasirox reverses hepatic fibrosis in β-thalassemia patients; however, smaller patient studies using either deferoxamine or deferiprone were unable to demonstrate a significant change in fibrosis score.27,28 While these studies suggest that iron chelation may lead to stabilization of hepatic fibrosis, deferasirox has been the only agent that has led to regression of fibrosis, highlighting the potential utility of this agent independent of its iron-chelating properties. We aimed to investigate the antifibrotic activity of deferasirox in both in vitro and in vivo models of hepatic fibrosis. The effect of deferasirox therapy on proliferation, viability, and fibrogenic gene expression in LX-2 cells was examined. Fifty micromolar deferasirox reduced the expression of α1(I)procollagen and αSMA after 12 h, and this reduction was maintained until 120 h. Reductions in MMP2, TIMP2, and TIMP1 were seen after 24, 48, and 96 h, respectively. Similar decreases in expression of these classic fibrogenic mediators were seen with 5 μM deferasirox, although these occurred later in the time-course. There was also a decrease in LX-2 proliferation following treatment with 50 μM deferasirox for 24 h, although this was not statistically significant. Surprisingly, there was not a commensurate reduction in TGF-β expression at this time. TGF-β mRNA was increased after 48 and 72 h; however, proliferation assays were not performed at these times. Jin et al.29 observed similar changes in rat primary HSCs following treatment with deferoxamine. Expression of αSMA, α2(I)procollagen, TIMP1, TIMP2, MMP-2, and MMP-9 mRNA was reduced in a dose-dependent manner. Iron supplementation abrogated the deferoxamine -induced reductions in αSMA and α2(I)procollagen, suggesting that the results can be attributed to iron chelation. Given the consistent results seen by Jin et al.29 and in the current study, it is possible that the antifibrotic changes in LX-2 642
cells are due to iron chelation, considering TfR1 expression was altered by deferasirox at later time points. It is, however, unclear whether the effect of deferasirox at early time points is independent of the amelioration of iron-induced oxidative stress as antioxidant gene expression was variable and did not show consistent downregulation throughout all time points studied. The current study also tested deferasirox as an antifibrotic agent in the Mdr2–/– mouse model of hepatic fibrosis. The dose of deferasirox used (30 mg/kg/day) has been shown to have limited effects on tissue iron concentration and enabled us to assess the antifibrotic activity of deferasirox independent of iron chelation.12 There were no changes in hepatic or splenic iron concentrations following deferasirox therapy and small increases in both serum iron and transferrin saturation, confirming that there was limited iron chelation at this dose. Despite reductions in fibrogenic gene expression in LX-2 cells, we were unable to demonstrate changes in serum transaminase levels, hepatic HP, Metavir fibrosis stage, ductular reaction score, or fibrogenic gene expression in Mdr2–/– mice following deferasirox therapy. One potential mechanism of action of deferasirox is via a reduction of NTBI, particularly the labile plasma iron.30 A sustained decrease of NTBI would likely lead to reduced oxidative damage and improvement of fibrosis. While this study did not address NTBI and labile plasma iron levels, the 4-week treatment period is likely to have been too short to observe changes that would result in decreased fibrosis. A longer duration of treatment and examination of NTBI may be an important avenue for future studies. Deugnier et al.9 described reductions in liver iron in their patient cohort; however, the antifibrotic effect was independent of this change in iron levels. Mdr2–/– mice have low hepatic iron levels in comparison with their wild-type controls (unpublished observations), and iron is not believed to play a role in the development of fibrosis in this model. This differs from the iron-induced fibrosis in transfusion-dependent β-thalassemia, and we cannot discount the possibility that the mechanism of deferasirox’s antifibrotic activity in patients would be different to those responsible for fibrosis development in our model.
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Figure 4 Deferasirox therapy does not reduce fibrosis in multidrug resistance 2 null (Mdr2–/–) mice. Mdr2–/– mice were treated with vehicle or deferasirox from 3 to 7 weeks of age and liver histology examined. (a) Hepatic fibrosis was quantified by hydroxyproline assay as described in the materials and methods section. (b–f) Liver histology was scored by a pathologist blinded to treatment groups using standard scoring systems. Representative liver sections from (g and i) vehicle and (h and j) deferasirox-treated mice stained with (g–h) Sirius red and (i–j) HE. Results are expressed as mean ± standard error of the mean (SEM), n = 6–7.
Kaji et al.31 have also demonstrated that deferasirox has antifibrotic activity in a rat model of non-alcoholic steatohepatitis. Both this study and the study by Deugnier et al.9 in β-thalassemia patients used high doses of deferasirox (up to 30 mg/kg/day in patients, 100 mg/kg/day in rats), and treatment occurred for a longer period of time (3.8–5.6 years in patients and 12 weeks in rats). Interestingly, analysis of the β-thalassemia cohort after 1
year of treatment did not demonstrate improvements in fibrosis.32 It is possible that the antifibrotic properties of deferasirox are only applicable at higher doses or that a longer period of treatment is required to see any effects. While this study has not confirmed the antifibrotic effects of deferasirox in the Mdr2–/– model we observed antifibrotic effects in an HSC line. Further investigation of deferasirox in other animal
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Figure 5 Expression of fibrogenic mediators in multidrug resistance 2 null (Mdr2–/–) mice following deferasirox therapy. Expression of (a) procollagen α1(I) (COL1A1), (b) transforming growth factor-β1 (Tgf-β), (c) platelet-derived growth factor receptor-B (Pdgfrb), and (d) α-smooth muscle actin (“α Sma”) mRNA was examined in livers of mice treated with vehicle or deferasirox from 3 to 7 weeks of age using real-time polymerase chain reaction (RT-PCR). Transcript levels were normalized to β2-microglobulin (β2-m) and basic transcription factor 3 (BTF3) levels. n = 6–7.
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Figure 6 Matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) expression as well as gelatinase activity are not altered following deferasirox therapy. Expression of (a) matrix metalloproteinase-2 (Mmp2), (b) tissue inhibitor of metalloproteinase-1 (Timp1) (b), and (c) Timp2 mRNA was examined using real-time polymerase chain reaction (RT-PCR). Transcript levels were normalized to β2-microglobulin (β2-m) and basic transcription factor 3 (BTF3) levels. (d) Gelatinase activity was assessed using zymography, (e and f) and zymograms were quantified by densitometry analysis. Results are expressed as mean ± standard error of the mean (SEM), n = 6–7.
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models at chelating and non-iron-chelating doses plus a longer duration of therapy will be required to further investigate the antifibrotic properties and mechanisms of action of deferasirox.
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Acknowledgments
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This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (APP1004517 and APP1024672), University of Queensland Reginald Ferguson Research Fellowship (KRB), Australian Postgraduate Award (ALS), and Gallipoli Medical Research Foundation scholarships (ALS, NS). LX-2 cells were a gift from Professor Scott Friedman.
References 1 Lindsey WT, Olin BR. Deferasirox for transfusion-related iron overload: a clinical review. Clin. Ther. 2007; 29: 2154–66. 2 Yang LP, Keam SJ, Keating GM. Deferasirox: a review of its use in the management of transfusional chronic iron overload. Drugs 2007; 67: 2211–30. 3 Kushner JP, Porter JP, Olivieri NF. Secondary iron overload. Hematology 2001; 1: 47–61. 4 Bacon BR, Britton RS. The pathology of hepatic iron overload: a free radical—mediated process? Hepatology 1990; 11: 127–37. 5 Piga A, Galanello R, Forni GL et al. Randomized phase II trial of deferasirox (Exjade, ICL670), a once-daily, orally-administered iron chelator, in comparison to deferoxamine in thalassemia patients with transfusional iron overload. Haematologica 2006; 91: 873–80. 6 Daar S, Pathare A, Nick H et al. Reduction in labile plasma iron during treatment with deferasirox, a once-daily oral iron chelator, in heavily iron-overloaded patients with beta-thalassaemia. Eur. J. Haematol. 2009; 82: 454–7. 7 Pennell DJ, Porter JB, Cappellini MD et al. Efficacy of deferasirox in reducing and preventing cardiac iron overload in beta-thalassemia. Blood 2010; 115: 2364–71. 8 Wood JC, Otto-Duessel M, Gonzalez I et al. Deferasirox and deferiprone remove cardiac iron in the iron-overloaded gerbil. Transl. Res. 2006; 148: 272–80. 9 Deugnier Y, Turlin B, Ropert M et al. Improvement in liver pathology of patients with beta-thalassemia treated with deferasirox for at least 3 years. Gastroenterology 2011; 141: 1202–11. 11 e1–3. 10 Popov Y, Patsenker E, Fickert P, Trauner M, Schuppan D. Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes. J. Hepatol. 2005; 43: 1045–54. 11 Xu L, Hui AY, Albanis E et al. Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 2005; 54: 142–51. 12 Nick H, Allegrini PR, Fozard L et al. Deferasirox reduces iron overload in a murine model of juvenile hemochromatosis. Exp. Biol. Med. 2009; 234: 492–503. 13 Tan TC, Crawford DH, Jaskowski LA et al. Altered lipid metabolism in Hfe-knockout mice promotes severe NAFLD and early fibrosis. Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 301: G865–76. 14 Jamall IS, Finelli VN, Que HSS. A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues. Anal. Biochem. 1981; 112: 70–5. 15 Jonsson JR, Clouston AD, Ando Y et al. Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis. Gastroenterology 2001; 121: 148–55. 16 Bridle KR, Sobbe AL, de Guzman CE et al. Lack of efficacy of mTOR inhibitors and ACE pathway inhibitors as antifibrotic agents
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in evolving and established fibrosis in Mdr2 mice. Liver Int. 2014; doi:10.1111/liv.12494. Richardson MM, Jonsson JR, Powell EE et al. Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction. Gastroenterology 2007; 133: 80–90. Clouston AD, Powell EE, Walsh MJ, Richardson MM, Demetris AJ, Jonsson JR. Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis. Hepatology 2005; 41: 809–18. Bridle K, Cheung TK, Murphy T et al. Hepcidin is down-regulated in alcoholic liver injury: implications for the pathogenesis of alcoholic liver disease. Alcohol. Clin. Exp. Res. 2006; 30: 106–12. Bridle KR, Popa C, Morgan ML et al. Rapamycin inhibits hepatic fibrosis in rats by attenuating multiple profibrogenic pathways. Liver Transpl. 2009; 15: 1315–24. Kossakowska AE, Edwards DR, Lee SS et al. Altered balance between matrix metalloproteinases and their inhibitors in experimental biliary fibrosis. Am. J. Pathol. 1998; 153: 1895–902. Gabrielsen JS, Gao Y, Simcox JA et al. Adipocyte iron regulates adiponectin and insulin sensitivity. J. Clin. Invest. 2012; 122: 3529–40. Shiratori Y, Imazeki F, Moriyama M et al. Histologic improvement of fibrosis in patients with hepatitis C who have sustained response to interferon therapy. Ann. Intern. Med. 2000; 132: 517–24. Farci P, Roskams T, Chessa L et al. Long-term benefit of interferon alpha therapy of chronic hepatitis D: regression of advanced hepatic fibrosis. Gastroenterology 2004; 126: 1740–9. Poynard T, McHutchison J, Manns M et al. Impact of pegylated interferon alfa-2b and ribavirin on liver fibrosis in patients with chronic hepatitis C. Gastroenterology 2002; 122: 1303–13. Falize L, Guillygomarc’h A, Perrin M et al. Reversibility of hepatic fibrosis in treated genetic hemochromatosis: a study of 36 cases. Hepatology 2006; 44: 472–7. Wanless IR, Sweeney G, Dhillon AP et al. Lack of progressive hepatic fibrosis during long-term therapy with deferiprone in subjects with transfusion-dependent beta-thalassemia. Blood 2002; 100: 1566–9. Berdoukas V, Bohane T, Tobias V et al. Liver iron concentration and fibrosis in a cohort of transfusion-dependent patients on long-term desferrioxamine therapy. Hematol. J. 2005; 5: 572–8. Jin H, Terai S, Sakaida I. The iron chelator deferoxamine causes activated hepatic stellate cells to become quiescent and to undergo apoptosis. J. Gastroenterol. 2007; 42: 475–84. List AF, Baer MR, Steensma DP et al. Deferasirox reduces serum ferritin and labile plasma iron in RBC transfusion-dependent patients with myelodysplastic syndrome. J. Clin. Oncol. 2012; 30: 2134–9. Kaji K, Yoshiji H, Kitade M et al. Combination treatment of angiotensin II type I receptor blocker and new oral iron chelator attenuates progression of nonalcoholic steatohepatitis in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 300: G1094–104. Brissot P, Turlin B, Forni GL et al. Iron chelation therapy with deferasirox (Exjade(R), ICL670) or deferoxamine results in reduced hepatocellular inflammation and improved liver function in patients with transfusion-dependent anemia. ASH Annu. Meet. Abstr. 2005; 106: 242A–3A.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1 Primer sequences and accession numbers for primers used for quantitative real-time polymerase chain reaction (qRTPCR)
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H E PAT O L O G Y
Inconsistent hepatic antifibrotic effects with the iron chelator deferasirox Amy Sobbe,* Kim R Bridle,* Lesley Jaskowski,* C Erika de Guzman,* Nishreen Santrampurwala,* Andrew D Clouston,† Catherine M Campbell,† V Nathan Subramaniam*,‡ and Darrell H G Crawford* *School of Medicine, The University of Queensland, Gallipoli Medical Research Foundation, Greenslopes Private Hospital, †Envoi Specialist Pathologists, and ‡Membrane Transport Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
Key words animal models, iron-chelating agents, liver fibrosis, stellate cells. Accepted for publication 17 August 2014. Correspondence Dr Kim R Bridle, School of Medicine, The University of Queensland, Lower Lobby Level, Administration Building, Greenslopes Private Hospital, Greenslopes, Qld 4120, Australia. Email: [email protected]
Abstract Background and Aim: Development of effective antifibrotic treatments that can be translated to clinical practice is an important challenge in contemporary hepatology. A recent report on β-thalassemia patients demonstrated that deferasirox treatment reversed or stabilized liver fibrosis independent of its iron-chelating properties. In this study, we investigated deferasirox in cell and animal models to better understand its potential antifibrotic effects. Methods: The LX-2 stellate cell line was treated with 5 μM or 50 μM deferasirox (Exjade, Novartis Pharmaceuticals Australia, North Ryde, NSW, Australia) for up to 120 h. Three-week-old multidrug resistance 2 null (Mdr2–/–) mice received oral deferasirox or vehicle for 4 weeks (30 mg/kg/day). Cells and liver tissue were collected for assessment of fibrosis and fibrogenic gene expression. Results: In LX-2 cells treated with 50 μM deferasirox for 12 h, α1(I)procollagen expression was decreased by 25%, with maximal reductions (10-fold) seen following 24–120 h of treatment. Similarly, α-smooth muscle actin (αSMA) expression was significantly lower. Alterations in matrix remodeling genes, specifically decreased expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2, were observed. There was no significant difference in hepatic hydroxyproline content in Mdr2–/– mice following deferasirox administration (vehicle: 395 ± 27 μg/g vs deferasirox: 421 ± 33 μg/g). Similarly, no changes in the expression of fibrogenic genes were observed. Conclusion: Despite reductions in α1(I)procollagen and αSMA expression and alterations in matrix degradation genes in LX-2 cells, deferasirox did not exhibit antifibrotic activity in Mdr2–/– mice. Given the positive outcomes seen in human trials, it may be appropriate to study deferasirox in other animal models of fibrosis and/or for a longer duration of therapy.
Introduction With the increasing prevalence of liver disease, antifibrotic therapies that can be translated to clinical practice are urgently needed. One agent that has shown beneficial effects on hepatic fibrosis in patients is deferasirox, an oral iron chelator used primarily for the management of transfusional iron overload in patients with chronic anemias (reviewed in Lindsey and Olin1 and Yang et al.2). In these patients, long-term transfusions result in accumulation of iron in the body, predominantly in the liver and heart.3 The consequences of excess hepatic iron include oxidative damage to cellular proteins and membrane lipids and, for many patients, hepatic fibrosis or cirrhosis.4 Deferasirox complexes with iron in a 2:1 (deferasirox : iron) ratio and is highly selective for Fe3+ over Fe2+.2 Chelation with 638
deferasirox reduces hepatic iron concentration, serum ferritin, non-transferrin-bound iron (NTBI)5,6 and reduces cardiac siderosis.7,8 However, Deugnier et al.9 have shown that deferasirox has antifibrotic activity independent of its iron-chelating properties. In β-thalassemia patients, treatment with deferasirox was associated with stability or improvement in liver fibrosis and a reduction in serum alanine aminotransferase (ALT) levels. In many patients, a reduction in Ishak fibrosis stage was seen without a reduction in hepatic iron concentration. Despite these favorable indications in patients, there have been limited studies investigating deferasirox as an antifibrotic agent in either cell culture or animal models of hepatic fibrosis that are not complicated by iron overload and the mechanisms responsible are largely unexplored. Multidrug resistance 2 null (Mdr2–/–) mice, a model of cholestatic liver disease, lack the cannalicular phospholipid flippase, and have significantly
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altered expression of fibrogenic genes by 4 weeks of age and fully developed fibrosis by 10 weeks of age.10 These mice provide an ideal model to study potential therapeutics for hepatic fibrosis. Similarly, the LX-2 cell (a human hepatic stellate cell [HSC] line)11 is an in vitro model suitable for studies on therapeutics for fibrosis. Thus, we investigated deferasirox therapy in LX-2 cells and in the Mdr2–/– mouse model of hepatic fibrosis to determine its potential antifibrotic activity.
Methods Cell culture. LX-2 cells11 were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Musgrave, Vic., Australia) supplemented with 10% fetal calf serum, 1% penicillin/ streptomycin, and 1% glutamax. Cells were cultured with deferasirox (Novartis Pharmaceuticals Australia, North Ryde, NSW, Australia) or vehicle (water) for 3, 6, 12, 24, 48, 72, 96, and 120 h.
Proliferation and viability. Cell proliferation was measured using a CellTiter96 Aqueous One Solution Cell Proliferation Assay (Promega, Alexandria, NSW, Australia), and cell viability was measured using a CellTiter-Blue Cell Viability Assay (Promega) as per the manufacturer’s instructions. Absorbance was measured using an Infinite F200 Plate Reader (Tecan, Port Melbourne, Vic., Australia).
Animals. Male and female Mdr2–/– mice (FVB-N background, The Jackson Laboratory, Bar Harbor, ME, USA) were used for these studies. Mice were kept under 12 h light/dark cycles and given ad libitum access to a standard mouse diet (Specialty Feeds, Glen Forrest, WA, Australia) and water. Mdr2–/– mice (four male and four female per group) were treated with vehicle (0.5% hydroxypropylcellulose) or deferasirox (30 mg/kg/day) by daily oral gavage from 3 to 7 weeks of age. This dose of deferasirox has previously been shown to have limited effects on tissue iron levels in a mouse model12 and allowed us to assess antifibrotic activity independent of iron chelation. The study was approved by the University of Queensland Animal Ethics Committee and performed according to the Australian code for the care and use of animals for scientific purposes.
Assessment of iron stores. Hepatic and splenic iron concentrations were measured as previously described.13 Serum iron, unsaturated iron-binding capacity, transferrin saturation, and total iron-binding capacity (TIBC) were measured with an Iron/TIBC Reagent Set (Pointe Scientific, Canton, MI, USA) according to the manufacturer’s instructions but scaled down to suit a 96-well plate format.
Serum biochemistry. Liver function tests were performed on serum to evaluate ALT, alkaline phosphatase (ALP) and bilirubin levels as per manufacturer’s instructions (Bioo Scientific, Austin, TX, USA).
Antifibrotic activity of deferasirox
Determination of hydroxyproline content. The hepatic concentration of hydroxyproline (HP) was measured using the method of Jamall et al.14 with modifications.15 HP levels were calculated from a standard curve of trans-4-hydroxy-L-proline run concurrently and expressed relative to tissue weight. Histological evaluation. Paraffin-embedded liver sections were used to quantify fibrosis, inflammation, and ductular reaction after HE and Sirius red staining. Sections were reviewed independently by two experienced histopathologists blinded to laboratory data and staged and graded according to accepted criteria and as published previously.16–18 RNA isolation and quantitative real-time polymerase chain reaction analysis. Isolation of total RNA was performed using TrizolTM Reagent (Life Technologies, Mulgrave, VIC, Australia) as per the manufacturer’s instructions and as previously described.19,20 Quantitative real-time polymerase chain reaction (PCR) using Sybr Green (Life Technologies) was performed on a Viia7 Real-Time PCR System (Life Technologies). Mouse and human primer nucleotide sequences and accession numbers can be found in Table S1. Results for each sample were calculated as a percentage of the β2-microglobulin (β2-m) and basic transcription factor 3 (BTF3) mRNA concentration (mouse samples) or of β2-microglobulin (β2-m) and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA concentration (LX-2 samples). Zymography. Ten micrograms of protein extract from liver was assayed for matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) activity by gelatin zymography as previously described.16,21 The relative activities of MMP-2 and MMP-9 for each sample were visualized using a Carestream Image Station 4000 MM Pro and quantified using Carestream Molecular Imaging software (Carestream, East Melbourne, Vic., Australia). Statistical analysis. Statistical analysis was performed using IBM SPSS Statistics 21 (IBM Corporation, New York, NY, USA). For in vitro studies at each timepoint, one-way anova analysis was performed. Where this global test indicated statistical significance, least significant differences post-hoc analysis was used to determine differences between treatment groups. For in vivo studies, differences between vehicle and deferasirox treated groups were assessed using a Students’ t-test. For all analyses, a P-value < 0.05 was considered statistically significant.
Results Effect of deferasirox on LX-2 cells. LX-2 cells were treated with 0, 5, or 50 μM deferasirox for 6 or 24 h. Proliferation and viability of cells was measured using the CellTiter96 Aqueous One Solution Cell Proliferation Assay and CellTiter-Blue Cell Viability Assay. No significant differences in LX-2 cell proliferation (P = 0.087 and 0.238) or viability (P = 0.194 and 0.664) were seen at either 6 or 24 h following deferasirox treatment (Fig. 1). To determine the effect of deferasirox on cellular iron status we measured transferrin receptor 1 (TfR1) expression, and to examine markers of oxidative stress we assessed gene expression of antioxidants glutathione peroxidase 1 (GPx1), manganese superoxide
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dismutase 2 (SOD2), and thioredoxin 1 (TXN). TfR1 expression has been used previously as a surrogate marker of cellular iron levels.22 Following 3–72 h deferasirox treatment, there were no significant changes in TfR1 expression compared with vehicle alone. Deferasirox treatment at 96 and 120 h resulted in increased TfR1 expression (96 h: 1.7-fold, P = 0.01 and 2.0-fold, P < 0.001 for 5 and 50 μM deferasirox, respectively, and 120 h: 1.8-fold, P = 0.02 and 1.3-fold, P = 0.05 for 5 and 50 μM deferasirox, respectively; results not shown). Following 5 μM deferasirox treatment, GPx1 expression was increased compared with vehicle at 24 h (1.2-fold, P = 0.002) and decreased at 48 h (0.9-fold, P = 0.03); SOD2 was decreased at 72, 96, and 120 h (0.8-fold, 0.7-fold, 0.6-fold; P = 0.01, 0.001, 0.02, respectively), and TXN gene expression was reduced at 48 and 120 h (0.9-fold and 0.8-fold, P = 0.03 and 0.03, respectively). Deferasirox treatment (50 μM) decreased GPx1 expression at 6–48 h (0.85-fold, 0.7-fold, 0.8-fold, 0.6-fold, P = 0.01, 0.01, 0.003, 0.001, respectively); SOD2 expression was increased at 6 and 24 h (1.2-fold and 1.2-fold, P = 0.01, 0.001, respectively) but decreased at 72 and 96 (0.8-fold, 0.7-fold, P = 0.01, 0.001 respectively), and TXN was reduced at 12, 24, and 48 h compared with vehicle (0.8-fold, 0.8-fold, 0.6-fold; P = 0.02, 0.002, 0.001, respectively; results not shown). Expression of the fibrogenic genes procollagen α1(I), (COL1A1), α-smooth muscle actin (αSMA), transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1, TIMP-2), and matrix metalloproteinase (MMP-2) were measured in LX-2 cells by qPCR at intervals from 12–120 h. COL1A1 and αSMA expression were significantly lower in LX-2 cells treated with 50 μM deferasirox for 12–120 h compared with untreated cells with maximal fold-reduction at 48 h treatment (11.5-fold and 8.3-fold, respectively; Figure 2a and c). Expression of COL1A1 and αSMA were also lower in LX-2 cells following 50 μM deferasirox compared with cells treated with 5 μM deferasirox with maximal fold-reduction seen at 48 h treatment (10.8-fold and 5.5-fold, respectively; Figure 2a and c). LX-2 cells treated with 5 μM deferasirox had lower COL1A1 and αSMA expression than untreated cells at 72, 96, and 120 h, respectively, with maximal reduction at 120 h (1.8-fold and 2.7-fold, respectively; Figure 2a and c). In contrast, at both 48 and 72 h, TGF-β1 expression was significantly higher in LX-2 cells treated with 50 μM deferasirox than in both untreated LX-2 cells (P = 0.012 and 0.003) and in those treated with 5 μM deferasirox (P = 0.002 and 0.002, Figure 2b). However, TIMP1 mRNA was significantly reduced by 50 μM deferasirox when compared with untreated cells at 96 h (1.4-fold, P = 0.011) and when compared with both 640
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Figure 1 Proliferation and viability in LX-2 cells treated with deferasirox. No significant differences were observed in LX-2 cell proliferation (a) nor LX-2 viability (b) at either 6 or 24 h following deferasirox treatment. Data are presented as the mean ± standard error of the mean (SEM) of duplicate determinations from three independent experiments. , 0 μM; , 5 μM; , 50 μM.
untreated cells and cells treated with 5 μM deferasirox at 120 h (1.4-fold, P = 0.001 and 1.2-fold, P = 0.006, respectively, Figure 2d). Similarly, TIMP2 expression was significantly reduced by 50 μM deferasirox when compared with untreated LX-2 cells after 48–120 h, with a maximal 2.6-fold-reduction at 120 h, and when compared with cells treated with 5 μM deferasirox at 48, 72, and 120 h (maximal 1.9-fold reduction at 120 h, Figure 2e). MMP2 expression was significantly lower in cells treated with 50 μM deferasirox than in untreated cells from 24–120 h treatment (P = 0.012, P = 0.001, P = 0.001, P < 0.001, and P < 0.001, respectively). MMP2 mRNA was significantly lower following 50 μM deferasirox treatment than in those treated with 5 μM deferasiox after 48 and 120 h (P = 0.018 and P = 0.001). Cells treated with 5 μM deferasirox had significantly lower MMP2 levels than untreated cells after 72, 96, and 120 h (P = 0.009, P = 0.002 and P < 0.001, respectively; Figure 2f). Body and liver weights, serum liver enzymes, and body iron stores of Mdr2–/– mice treated with deferasirox. Mdr2–/– mice were treated with deferasirox from 3 to 7 weeks of age. No significant differences in body weight (24.8 ± 1.0 vs 24.1 ± 0.8 g, P = 0.59), liver weight (2.4 ± 0.1 vs 2.3 ± 0.1 g, P = 0.56), or the liver to body weight ratio (9.6 ± 0.4 vs 9.5 ± 0.2%, P = 0.76) in Mdr2–/– mice was observed when compared with vehicle-treated animals. Similarly, there were no significant differences in serum ALT (364 ± 45 vs 361 ± 49 IU/L, P = 0.97), ALP (46 ± 4 vs 61 ± 8 IU/L, P = 0.13), or bilirubin (2.7 ± 0.7 vs 3.5 ± 0.7 g/dL, P = 0.43) levels between vehicle and deferasirox-treated groups (Table 1). Serum iron and transferrin saturation were significantly increased in Mdr2–/– mice following deferasirox treatment (300 ± 16 vs 234 ± 16 μg/dL, P = 0.015 and 39.8 ± 0.6 vs 33.2 ± 1.5%, P = 0.006, respectively; Figure 3a and b). In contrast, deferasirox treatment did not significantly alter either hepatic or splenic iron concentration (17.2 ± 1.8 vs 15.1 ± 2.0 μmol Fe/g dry weight, P = 0.478 and 71.1 ± 3.5 vs 61.1 ± 6.2 μmol Fe/g dry weight, P = 0.203, respectively; Figure 3c and d). Expression of antioxidant genes GPx1, SOD2, and TXN were not significantly different in animals treated with deferasirox compared with those given vehicle alone (0.92-fold, P = 0.6; 0.86-fold, P = 0.05; 0.9fold, P = 0.1; respectively; results not shown). Assessment of injury and fibrosis in Mdr2–/– treated with deferasirox for 4 weeks. Hepatic HP levels were similar in vehicle and deferasirox treated Mdr2–/– mice
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Figure 2 Expression of fibrogenic genes is reduced in LX-2 cells treated with deferasirox. LX-2 cells were treated with ) 0, ( ) 5, or ( ) 50 μM deferasirox for ( 3–120 h. Real-time polymerase chain reaction (RT-PCR) was used to analyze expression of (a) procollagen α1(I) (COL1A1), (b) transforming growth factor-β (TGF-β), (c) α-smooth muscle actin (αSMA), (d) tissue inhibitors of metalloproteinase-1 (TIMP1), (e) TIMP2, and (f) matrix metalloproteinase-2 MMP2. Transcript levels were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) and basic transcription factor 3 (BTF3) expression. Data are presented as the mean ± standard error of the mean (SEM) of duplicate determinations from two or three independent experiments at each time-point. *P < 0.05 50 μM versus 0 μM deferasirox, #P < 0.05 5 μM versus 0 μM deferasirox, ~P < 0.05 50 μM versus 5 μM deferasirox.
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24.8 ± 1.0 2.4 ± 0.1 9.6 ± 0.4 364 ± 45 46 ± 4 2.7 ± 0.7
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n = 6–7. Body and liver weights were recorded at the end of the 4-week treatment period. Liver weight was expressed as a percentage of body weight. Serum liver enzymes were measured using commercial assays according to the manufacturer’s instructions. Results are expressed as mean ± standard error of the mean (SEM). ALP, alkaline phosphatase; ALT, alanine transaminase; Mdr2–/–, multidrug resistance 2 null; NS, not significant.
(395 ± 27 vs 421 ± 33 μg/g, P = 0.57, Figure 4a). Similarly, Metavir fibrosis stage (3.2 ± 0.3 vs 3.0 ± 0, P = 0.61, Figure 4b), ductular reaction score (3.8 ± 0.2 vs 4.0 0.0, P = 0.36, Figure 4c), portal inflammation (3.2 ± 0.4 vs 3.4 ± 0.2, P = 0.58, Figure 4d), and lobular inflammation (1.8 ± 0.2 vs 2.0 ± 0.4, P = 0.71, Figure 4e) and hepatocyte ballooning (1.2 ± 0.3 vs 0.6 ± 0.2, P = 0.12, Figure 4f) were not reduced following deferasirox treat-
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ment from 3 to 7 weeks of age. Representative liver sections stained with Sirius red and HE are shown in Figure 4g–j.
Fibrogenic gene expression and matrix remodelling in livers of Mdr2–/– mice treated with deferasirox. Expression of Col1a1, Tgf-β, and plateletderived growth factor receptor-B (Pdgfrb) mRNA was not significantly altered following deferasirox treatment from 3 to 7 weeks of age (P = 0.39, 0.29, and 0.71 respectively; Figure 5a–c). In contrast, αSma was increased 1.9-fold in Mdr2–/– mice following deferasirox therapy from 3 to 7 weeks of age; however, this did not reach statistical significance (P = 0.26; Figure 5d). No significant differences in Mmp2, Timp1, or Timp2 expression were noted following deferasirox treatment (P = 0.51, 0.32, and 0.68 respectively; Figure 6a–c). Similarly, gelatinase (MMP2 and MMP9) activity was not significantly different between vehicle and deferasirox-treated mice (P = 0.48 and 0.88 respectively; Figure 6d–f).
Discussion Reversal of hepatic fibrosis has been demonstrated with a variety of therapies and in injury of various aetiologies. For example, histological improvement has been observed in patients with hepa-
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Figure 3 Deferasirox treatment does not reduce serum, hepatic, and splenic iron levels in multidrug resistance 2 null (Mdr2–/–) mice. (a) Serum iron, (b) transferrin saturation, and (c) hepatic and (d) splenic iron concentrations were measured in Mdr2–/– mice treated with vehicle or deferasirox from 3 to 7 weeks of age. Results are expressed as mean ± standard error of the mean (SEM). n = 6–7. *P < 0.05 vehicle versus deferasirox.
titis C and D who have sustained response to interferon therapy23–25 and in patients who have received long-term venesection for hemochromatosis.26 These therapies primarily target the cause of injury whereas agents that inhibit the fibrogenic process have shown little efficacy in patients. Deugnier et al.9 have demonstrated that deferasirox reverses hepatic fibrosis in β-thalassemia patients; however, smaller patient studies using either deferoxamine or deferiprone were unable to demonstrate a significant change in fibrosis score.27,28 While these studies suggest that iron chelation may lead to stabilization of hepatic fibrosis, deferasirox has been the only agent that has led to regression of fibrosis, highlighting the potential utility of this agent independent of its iron-chelating properties. We aimed to investigate the antifibrotic activity of deferasirox in both in vitro and in vivo models of hepatic fibrosis. The effect of deferasirox therapy on proliferation, viability, and fibrogenic gene expression in LX-2 cells was examined. Fifty micromolar deferasirox reduced the expression of α1(I)procollagen and αSMA after 12 h, and this reduction was maintained until 120 h. Reductions in MMP2, TIMP2, and TIMP1 were seen after 24, 48, and 96 h, respectively. Similar decreases in expression of these classic fibrogenic mediators were seen with 5 μM deferasirox, although these occurred later in the time-course. There was also a decrease in LX-2 proliferation following treatment with 50 μM deferasirox for 24 h, although this was not statistically significant. Surprisingly, there was not a commensurate reduction in TGF-β expression at this time. TGF-β mRNA was increased after 48 and 72 h; however, proliferation assays were not performed at these times. Jin et al.29 observed similar changes in rat primary HSCs following treatment with deferoxamine. Expression of αSMA, α2(I)procollagen, TIMP1, TIMP2, MMP-2, and MMP-9 mRNA was reduced in a dose-dependent manner. Iron supplementation abrogated the deferoxamine -induced reductions in αSMA and α2(I)procollagen, suggesting that the results can be attributed to iron chelation. Given the consistent results seen by Jin et al.29 and in the current study, it is possible that the antifibrotic changes in LX-2 642
cells are due to iron chelation, considering TfR1 expression was altered by deferasirox at later time points. It is, however, unclear whether the effect of deferasirox at early time points is independent of the amelioration of iron-induced oxidative stress as antioxidant gene expression was variable and did not show consistent downregulation throughout all time points studied. The current study also tested deferasirox as an antifibrotic agent in the Mdr2–/– mouse model of hepatic fibrosis. The dose of deferasirox used (30 mg/kg/day) has been shown to have limited effects on tissue iron concentration and enabled us to assess the antifibrotic activity of deferasirox independent of iron chelation.12 There were no changes in hepatic or splenic iron concentrations following deferasirox therapy and small increases in both serum iron and transferrin saturation, confirming that there was limited iron chelation at this dose. Despite reductions in fibrogenic gene expression in LX-2 cells, we were unable to demonstrate changes in serum transaminase levels, hepatic HP, Metavir fibrosis stage, ductular reaction score, or fibrogenic gene expression in Mdr2–/– mice following deferasirox therapy. One potential mechanism of action of deferasirox is via a reduction of NTBI, particularly the labile plasma iron.30 A sustained decrease of NTBI would likely lead to reduced oxidative damage and improvement of fibrosis. While this study did not address NTBI and labile plasma iron levels, the 4-week treatment period is likely to have been too short to observe changes that would result in decreased fibrosis. A longer duration of treatment and examination of NTBI may be an important avenue for future studies. Deugnier et al.9 described reductions in liver iron in their patient cohort; however, the antifibrotic effect was independent of this change in iron levels. Mdr2–/– mice have low hepatic iron levels in comparison with their wild-type controls (unpublished observations), and iron is not believed to play a role in the development of fibrosis in this model. This differs from the iron-induced fibrosis in transfusion-dependent β-thalassemia, and we cannot discount the possibility that the mechanism of deferasirox’s antifibrotic activity in patients would be different to those responsible for fibrosis development in our model.
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Figure 4 Deferasirox therapy does not reduce fibrosis in multidrug resistance 2 null (Mdr2–/–) mice. Mdr2–/– mice were treated with vehicle or deferasirox from 3 to 7 weeks of age and liver histology examined. (a) Hepatic fibrosis was quantified by hydroxyproline assay as described in the materials and methods section. (b–f) Liver histology was scored by a pathologist blinded to treatment groups using standard scoring systems. Representative liver sections from (g and i) vehicle and (h and j) deferasirox-treated mice stained with (g–h) Sirius red and (i–j) HE. Results are expressed as mean ± standard error of the mean (SEM), n = 6–7.
Kaji et al.31 have also demonstrated that deferasirox has antifibrotic activity in a rat model of non-alcoholic steatohepatitis. Both this study and the study by Deugnier et al.9 in β-thalassemia patients used high doses of deferasirox (up to 30 mg/kg/day in patients, 100 mg/kg/day in rats), and treatment occurred for a longer period of time (3.8–5.6 years in patients and 12 weeks in rats). Interestingly, analysis of the β-thalassemia cohort after 1
year of treatment did not demonstrate improvements in fibrosis.32 It is possible that the antifibrotic properties of deferasirox are only applicable at higher doses or that a longer period of treatment is required to see any effects. While this study has not confirmed the antifibrotic effects of deferasirox in the Mdr2–/– model we observed antifibrotic effects in an HSC line. Further investigation of deferasirox in other animal
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Figure 5 Expression of fibrogenic mediators in multidrug resistance 2 null (Mdr2–/–) mice following deferasirox therapy. Expression of (a) procollagen α1(I) (COL1A1), (b) transforming growth factor-β1 (Tgf-β), (c) platelet-derived growth factor receptor-B (Pdgfrb), and (d) α-smooth muscle actin (“α Sma”) mRNA was examined in livers of mice treated with vehicle or deferasirox from 3 to 7 weeks of age using real-time polymerase chain reaction (RT-PCR). Transcript levels were normalized to β2-microglobulin (β2-m) and basic transcription factor 3 (BTF3) levels. n = 6–7.
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models at chelating and non-iron-chelating doses plus a longer duration of therapy will be required to further investigate the antifibrotic properties and mechanisms of action of deferasirox.
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Acknowledgments
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This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (APP1004517 and APP1024672), University of Queensland Reginald Ferguson Research Fellowship (KRB), Australian Postgraduate Award (ALS), and Gallipoli Medical Research Foundation scholarships (ALS, NS). LX-2 cells were a gift from Professor Scott Friedman.
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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1 Primer sequences and accession numbers for primers used for quantitative real-time polymerase chain reaction (qRTPCR)
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