Treatment with imatinib prevents fibrosis in different preclinical models of systemic sclerosis and induces regression of established fibrosis

Authors

  • Alfiya Akhmetshina,

    1. University of Erlangen–Nuremberg, Erlangen, Germany
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  • Paulius Venalis,

    1. University of Erlangen–Nuremberg, Erlangen, Germany
    2. Vilnius University, Vilnius, Lithuania
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  • Clara Dees,

    1. University of Erlangen–Nuremberg, Erlangen, Germany
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  • Nicole Busch,

    1. University of Erlangen–Nuremberg, Erlangen, Germany
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  • Jochen Zwerina,

    1. University of Erlangen–Nuremberg, Erlangen, Germany
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  • Georg Schett,

    1. University of Erlangen–Nuremberg, Erlangen, Germany
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  • Oliver Distler,

    1. University Hospital Zurich, Zurich, Switzerland
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    • Drs. Oliver Distler and Jörg H. W. Distler have received consulting fees, speaking fees, and/or honoraria from Encysive and Actelion (less than $10,000 each). They also have received support from Novartis for a clinical trial with imatinib in systemic sclerosis.

  • Jörg H. W. Distler

    Corresponding author
    1. University of Erlangen–Nuremberg, Erlangen, Germany
    • Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen–Nuremberg, Universitätsstrasse 29, 91054 Erlangen, Germany
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    • Drs. Oliver Distler and Jörg H. W. Distler have received consulting fees, speaking fees, and/or honoraria from Encysive and Actelion (less than $10,000 each). They also have received support from Novartis for a clinical trial with imatinib in systemic sclerosis.


Abstract

Objective

Imatinib is a small-molecule tyrosine kinase inhibitor capable of selective, dual inhibition of the transforming growth factor β and platelet-derived growth factor (PDGF) pathways. Imatinib has previously been shown to prevent the development of inflammation-driven experimental fibrosis when treatment was initiated before administration of the profibrotic stimulus. The aim of this study was to confirm the efficacy of imatinib in a murine model of systemic sclerosis (SSc) that is less driven by inflammation and to investigate whether imatinib is also effective for the treatment of established fibrosis.

Methods

The tight skin 1 (TSK-1) mouse model of SSc was used to evaluate the antifibrotic effects of imatinib in a genetic model of the later stages of SSc. In addition, the efficacy of imatinib for the treatment of preestablished fibrosis was analyzed in a modified model of bleomycin-induced dermal fibrosis in which the application of bleomycin was prolonged and the onset of treatment was late.

Results

Treatment with imatinib reduced dermal and hypodermal thickening in TSK-1 mice and prevented the differentiation of resting fibroblasts into myofibroblasts. In the model of preestablished dermal fibrosis, imatinib not only stopped further progression of fibrosis but also induced regression of preexisting dermal fibrosis, with a reduction in dermal thickness below pretreatment levels.

Conclusion

These results indicate that combined inhibition of the tyrosine kinase c-Abl and PDGF receptor might be effective in the later, less inflammatory stages of SSc and for the treatment of established fibrosis. Thus, imatinib might be an interesting candidate for clinical trials in patients with longstanding disease and preexisting tissue fibrosis.

Systemic sclerosis (SSc) is a chronic fibrotic disorder of unknown etiology that affects the skin and a variety of internal organs. During the course of the disease, there is excessive accumulation of extracellular matrix (ECM) components in the skin and involved organs (1). The resulting fibrosis disrupts the physiologic structure of the affected tissues and can lead to severe dysfunction of the involved organs. Tissue fibrosis not only is a major cause of morbidity in SSc but also contributes significantly to the increased mortality of SSc patients (2). The accumulation of ECM in SSc is mediated by activated fibroblasts, which produce increased amounts of ECM proteins (3). Transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF) are considered to play important roles in fibroblast activation in SSc (3).

Imatinib mesylate (Gleevec/Glivec; Novartis, Basel, Switzerland) is an orally administered drug that is widely used for the treatment of Bcr/Abl–positive chronic myelogenous leukemia and gastrointestinal stromal tumors. Previous clinical trials in patients with chronic myelogenous leukemia demonstrated that imatinib is relatively well tolerated (4). Imatinib targets specifically the TGFβ and PDGF signaling pathways by inhibiting the tyrosine kinase activity of c-Abl and PDGF receptors and thus interferes simultaneously with 2 major pathways for the activation of fibroblasts in SSc (5). Recently, we demonstrated that imatinib prevented the development of dermal fibrosis upon challenge with bleomycin, when the treatment was initiated before the first injection of bleomycin (6). Imatinib has also been shown to be preventive in inflammatory models of fibrosis in other organs (7–9).

In SSc, prominent tissue inflammation is mainly restricted to very early stages of the disease and rarely occurs in the setting of longstanding disease. Thus, results obtained from these models of inflammatory tissue fibrosis mimic the early stages of SSc but are less representative of the later stages of SSc. Another limitation of previous studies is that they analyzed only whether imatinib can prevent the development of fibrosis. However, most patients with diffuse SSc are seen by the rheumatologist when significant tissue fibrosis has already occurred. Thus, the aim of therapy for such patients would be to stop disease progression and even induce regression of preexisting fibrosis. Ideal antifibrotic drugs should sufficiently decrease the production of collagen to shift the balance between matrix synthesis and matrix degradation toward matrix degradation, with a subsequent reduction in the preexisting accumulation of ECM. Kay and High recently reported that imatinib induced regression of the skin thickness score in 2 patients with nephrogenic systemic fibrosis (10). Considering the potent antifibrotic effects of imatinib on the prevention of fibrosis in models of inflammation and the findings by Kay and High, we hypothesized that imatinib might also be effective in fibrosis models mimicking longstanding disease and for the treatment of preestablished tissue fibrosis.

MATERIALS AND METHODS

Prevention of fibrosis in TSK-1 mice by imatinib.

To confirm the efficacy of imatinib in a model of SSc that is less dependent on inflammatory changes than is bleomycin-induced dermal fibrosis, the antifibrotic potential of imatinib was evaluated in the TSK-1 mouse model of SSc. Due to a dominant mutation of the fibrillin 1 gene, the phenotype of tsk1 is characterized by increased dermal and hypodermal thickness (11, 12). TSK-1 mice were interbred with pa/pa mice, in which a recessive mutation (pa) induces a light grey color of the fur and pink eyes. Because the fibrillin 1 gene is genetically linked to the pa gene, mice can be prescreened for the tsk1 mutation based on the color of their fur and eyes. All mice with black fur and eyes carry the dominant tsk1 mutation and are heterozygous for the pale mutation. In contrast, mice with light grey fur do not carry the tsk1 mutation but are homozygous for the mutated pale gene. Apart from the change in skin color, the pale mutation itself does not alter skin physiology or fibrogenesis.

Imatinib was dissolved in 0.9% NaCl and injected intraperitoneally in a total volume of 100 μl. Three groups of mice (total of 27) were analyzed. The injection scheme is presented in Figure 1a. One group of TSK-1 mice received imatinib at a dosage of 150 mg/kg/day, while another group of TSK-1 mice received an injection of the solvent NaCl. The third group consisted of pa/pa mice on the same genetic background not carrying the tsk1 mutation (controls); these mice also received intraperitoneal injections of NaCl. Treatment was started at age 5 weeks. After 5 weeks of treatment, mice were killed by cervical dislocation, and the skin was processed further for histologic analysis.

Figure 1.

Experimental design for imatinib treatment in TSK-1 mice (a) and mice with bleomycin-induced, established dermal fibrosis (b). Daggers indicate the time at which the mice were killed.

Imatinib treatment of established bleomycin-induced dermal fibrosis.

Skin fibrosis was induced in 6-week-old DBA mice by local intracutaneous injections of 100 μl of bleomycin dissolved in 0.9% NaCl, at a concentration of 0.5 mg/ml, every other day in defined areas of 1.5 cm2 on the upper back. The injection schemes for the 6 different groups are presented in Figure 1b. Briefly, one group of mice were killed after 3 weeks of treatment with bleomycin to analyze the fibrotic changes before treatment with imatinib. Another group of mice were killed after receiving bleomycin injections for 6 weeks. The third group received bleomycin injections for 3 weeks followed by NaCl injections for the next 3 weeks, to control for spontaneous regression of fibrosis. To assess the effects of imatinib in established fibrosis, mice were challenged with bleomycin for 6 weeks and treated in parallel with imatinib at dosages of 150 mg/kg/day for the last 3 weeks. Two groups of mice receiving intracutaneous injections of 100 μl 0.9% NaCl for 3 weeks and 6 weeks, respectively, were used as controls. All mice that were not treated with imatinib received intraperitoneal injections of NaCl. A total of 49 mice were analyzed.

Histologic analysis.

Skin sections were stained with hemalaun/eosin for better visualization of the tissue structure. Dermal thickness was analyzed with a Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, The Netherlands) by measuring the maximal distance between the epidermal–dermal junction and the dermal–subcutaneous fat junction at 4 different skin sections in each mouse, as previously described (13). Hypodermal thickness was determined by measuring the thickness of the subcutaneous connective tissue beneath the panniculus carnosus at 4 different sites of the upper back in each mouse. The evaluation was performed by 2 independent examiners.

Detection of myofibroblasts.

For quantification of myofibroblasts, skin sections were deparaffinized and incubated with 5% bovine serum albumin for 60 minutes. Cells positive for α-smooth muscle actin (α-SMA) were detected by incubation with monoclonal anti–α-SMA antibodies (clone 1A4; Sigma-Aldrich, Steinheim, Germany) for 2 hours at room temperature followed by incubation with 3% hydrogen peroxide for 10 minutes. Goat anti-rabbit antibodies labeled with horseradish peroxidase (Dako, Hamburg, Germany) were used as secondary antibodies. The expression of α-SMA was visualized with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). Monoclonal mouse IgG antibodies (Calbiochem, San Diego, CA) were used as controls (14).

Statistical analysis.

Data are expressed as the mean ± SD. The Mann-Whitney U test was used for statistical analyses. P values less than 0.05 were considered significant.

RESULTS

Correction of the TSK-1 phenotype by imatinib.

We first aimed to assess whether imatinib is effective in an in vivo model of dermal fibrosis that is largely independent from inflammation. Thus, we evaluated the efficacy of imatinib in the TSK-1 mouse model. TSK-1 mice are characterized by a modest increase in dermal thickness and strongly increased hypodermal thickness. Treatment with imatinib almost completely prevented these histologic changes in TSK-1 mice (Figures 2a–d). Dermal thickness was increased in TSK-1 mice by 40 ± 6% (mean ± SD) compared with pa/pa mice (P = 0.003). Treatment with imatinib reduced dermal thickness to normal levels (−5% ± 8%; P = 0.005) (Figures 2a and b). The increased thickness of the hypodermis in TSK-1 mice was also significantly reduced by imatinib, from 298 ± 9% to 84 ± 6% (P = 0.005) (Figures 2a and c).

Figure 2.

Antifibrotic effects of imatinib in TSK-1 mice. a, Dermal and hypodermal thickening in TSK-1 mice treated with imatinib was significantly reduced compared with that in mock-treated TSK-1 mice. Representative sections are shown. Top vertical bars show dermal thickness; lower vertical bars show hypodermal thickness (original magnification × 40). b, Imatinib treatment induced reduction of dermal thickness in TSK-1 mice. c, Hypodermal thickness was decreased in TSK-1 mice treated with imatinib. d, Myofibroblast differentiation was prevented by imatinib. Values in b–d are the mean and SD.

Myofibroblasts are considered as major effector cells for fibrosis. Imatinib significantly reduced the differentiation of resting fibroblasts into myofibroblasts in TSK-1 mice (Figure 2d). The number of myofibroblasts was reduced from 304 ± 28% in untreated TSK-1 mice to normal in TSK-1 mice treated with imatinib (−10 ± 10%; P = 0.0001).

Of note, treatment with imatinib completely normalized dermal thickness and myofibroblast counts. The mean dermal thickness and the numbers of myofibroblasts did not differ between control mice and TSK-1 mice treated with imatinib (P = 0.34 and P = 0.85, respectively).

Imatinib-induced regression of established fibrosis.

Although the prevention of fibrosis is a major aim in the treatment of SSc, the clinical situation is most often characterized by patients who present with already-established fibrosis. Thus, potential antifibrotic drugs for SSc not only should prevent fibrosis but also should induce regression of preexisting tissue fibrosis. To evaluate the efficacy of imatinib for the treatment of established fibrosis, we used a modified model of bleomycin-induced dermal fibrosis.

Consistent with previous studies, dermal thickness increased by 50 ± 2% (mean ± SD) after 3 weeks of treatment with bleomycin (P = 0.002 versus controls) (data not shown). Prolonged administration of bleomycin further increased dermal thickness. When the challenge with bleomycin was continued for an additional 3 weeks (total of 6 weeks), dermal thickness increased by 68 ± 3% (P = 0.001 versus 3 weeks and P = 0.008 versus controls). Treatment with imatinib for the last 3 weeks of bleomycin challenge not only stopped further progression of fibrosis but also induced regression of preexisting ECM accumulation and decreased dermal thickness below pretreatment levels (Figures 3a and b). Dermal thickness in mice treated with imatinib for the last 3 weeks was significantly reduced to 24 ± 4% compared with that in mice injected with bleomycin for 6 weeks (P = 0.002). Dermal thickness in mice injected with bleomycin for 6 weeks and treated with imatinib for the last 3 weeks was also significantly lower than that in mice challenged with bleomycin for 3 weeks (P = 0.003). These findings suggest that imatinib not only prevents the development of fibrosis but also can induce regression of preexisting fibrotic damage.

Figure 3.

Imatinib-induced regression of preexisting skin fibrosis in a modified model of bleomycin-induced fibrosis. a, Treatment with imatinib not only prevented further matrix accumulation but also induced regression of preexisting fibrosis. Representative sections are shown (original magnification × 100). b, Treatment with imatinib decreased dermal thickness to below baseline levels in mice with bleomycin-induced dermal fibrosis. Values are the mean and SD.

DISCUSSION

We have previously shown that imatinib prevents the development of fibrosis in the mouse model of bleomycin-induced dermal fibrosis (6). This model of dermal fibrosis is characterized by dense inflammatory infiltrates in lesional skin. Inflammatory cells are thought to contribute to the initial activation of resident fibroblasts by the release of profibrotic mediators. Thus, the mouse model of bleomycin-induced dermal fibrosis mimics early stages of SSc but is less representative for later stages of SSc, when inflammatory infiltrates are scarce (3). To evaluate the antifibrotic efficacy of imatinib in a model for later stages of SSc, we used the TSK-1 mouse model, in which persistent overproduction of ECM proteins occurs in the absence of inflammatory infiltrates (12). We demonstrate potent antifibrotic effects of imatinib in TSK-1 mice with prevention of histologic changes and inhibition of myofibroblast differentiation. Thus, our data obtained in TSK-1 mice indicate that imatinib might be effective not only for prevention of fibrosis in early, inflammatory stages of SSc but also in later stages of SSc, when inflammation is no longer dominant.

Regression of established fibrosis upon treatment with imatinib is probably mediated by a relative increase of matrix degradation relative to neosynthesis of ECM components. In our previous studies (6, 13), we demonstrated that combined inhibition of c-Abl and PDGF receptor decreases the synthesis of collagen by up to 80%. Direct effects of imatinib on matrix-degrading enzymes or their inhibitors were not observed. Thus, imatinib might induce regression of fibrosis via its potent inhibitory effects on collagen synthesis, leading to a relative increase in matrix degradation, rather than by exerting direct effects on matrix degradation.

The data obtained in TSK-1 mice in the present study and in the model of acute bleomycin-induced dermal fibrosis in our previous study (6) demonstrate that imatinib effectively prevents the development of dermal fibrosis in different preclinical models of SSc. In clinical practice, the prevention of fibrosis is mainly relevant for patients with very early, progressive SSc who are at high risk of significant morbidity due to the progressive deposition of ECM proteins in different organs. In contrast, the majority of patients present to rheumatology clinics with some degree of established fibrosis. Thus, treatment of established fibrosis is a frequent and urgent aim in this disease. In these patients, treatment would remove preexisting fibrotic changes and potentially reduce organ dysfunction.

Using a modified model of bleomycin-induced dermal fibrosis in which the application of bleomycin was prolonged and the onset of treatment was late, we demonstrate that imatinib not only halts progression of fibrosis but even reduces preexisting dermal fibrosis and decreases dermal thickness below pretreatment levels despite ongoing challenge with the profibrosis stimulus bleomycin. Thus, imatinib not only might stop progression but also might induce regression of tissue fibrosis. Imatinib might therefore also be an interesting candidate for clinical trials in SSc patients with extensive, longstanding disease and preexisting fibrotic organ damage.

However, potential antiangiogenic adverse effects of imatinib are an important concern, particularly in patients with SSc who have severe microvascular disease and recurrent ulcers. Although imatinib might not affect endothelial cells directly (15), careful monitoring of patients with SSc for exacerbation of vascular disease is warranted in clinical trials with imatinib.

Taken together, our results show that imatinib exerted potent antifibrotic effects in 2 in vivo models of SSc with different underlying pathologic mechanisms. Imatinib was effective for the prevention of fibrosis and the treatment of established dermal fibrosis. Furthermore, imatinib also exerted potent antifibrotic effects in preclinical models of other fibrotic diseases (7–9). The results of the present study indicate that combined inhibition of c-Abl and PDGF receptor might be effective for the treatment of established fibrosis as well. These findings enhance our excitement about the upcoming results of clinical trials with imatinib in SSc and other fibrotic disorders. The results from these trials, including their safety analyses, must be awaited before imatinib can be used routinely in the daily clinical care of patients with SSc.

AUTHOR CONTRIBUTIONS

Dr. J. Distler had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Akhmetshina, Schett, O. Distler, J. Distler.

Acquisition of data. Akhmetshina, Venalis, Dees, Busch, Zwerina, J. Distler.

Analysis and interpretation of data. Akhmetshina, Venalis, Busch, Schett, O. Distler, J. Distler.

Manuscript preparation. Akhmetshina, Schett, O. Distler, J. Dislter.

Statistical analysis. Akhmetshina, J. Distler.

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