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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Tissue fibrosis caused by pathologic activation of fibroblasts with increased synthesis of extracellular matrix components is a major hallmark of systemic sclerosis (SSc). Notch signaling regulates tissue differentiation, and abnormal activation of Notch signaling has been implicated in the pathogenesis of various malignancies. The present study was undertaken to investigate the role of Notch signaling in SSc and to evaluate the therapeutic potential of Notch inhibition for the treatment of fibrosis.

Methods

Activation of the Notch pathways was analyzed by staining for the Notch intracellular domain (NICD) and quantification of levels of HES-1 messenger RNA. In the mouse model of bleomycin-induced dermal fibrosis and in tight skin 1 mice, Notch signaling was inhibited by the γ-secretase inhibitor DAPT and by overexpression of a Notch-1 antisense construct.

Results

Notch signaling was activated in SSc in vivo, with accumulation of the NICD and increased transcription of the target gene HES-1. Overexpression of a Notch antisense construct prevented bleomycin-induced fibrosis and hypodermal thickening in tight skin 1 mice. Potent antifibrotic effects were also obtained with DAPT treatment. In addition to prevention of fibrosis, targeting of Notch signaling resulted in almost complete regression of established experimental fibrosis.

Conclusion

The present results demonstrate that pharmacologic as well as genetic inhibition of Notch signaling exerts potent antifibrotic effects in different murine models of SSc. These findings might have direct translational implications because different inhibitors of the γ-secretase complex are available and have yielded promising results in cancer trials.

Systemic sclerosis (SSc) is a chronic fibrotic disease of unknown etiology that affects the skin and several internal organs, including the lungs, heart, gastrointestinal tract, and kidneys. Early stages of SSc are characterized by activation of the immune system and infiltration of affected tissue with leukocytes (1). The infiltrating leukocytes, primarily T cells, monocytes, and B cells, release profibrotic mediators that may initially drive the activation of resident fibroblasts. However, the activation of SSc fibroblasts persists even after the inflammatory infiltration has resolved, suggesting an endogenous activation (2). The excessive release of collagen by SSc fibroblasts leads to progressive fibrosis and is a major cause of death in SSc (3). Treatment of tissue fibrosis represents a particular challenge in disease management. Current approaches to treat fibrosis are of limited efficacy, and therapies specifically targeting the activation of fibroblasts and the release of extracellular matrix are not available for clinical use to date (4–6).

The Notch gene was first discovered in Drosophila mutants, which developed notches at the tips of their wing blades (7). The Notch gene transcribes into receptors consisting of one large transmembrane domain and is evolutionarily conserved (8). Binding of ligands such as Jag-1 to the Notch receptor induces cleavage of Notch at 2 different sites. The cleavage step catalyzed by the γ-secretase complex results in the release of the Notch intracellular domain (NICD) (8). The NICD then translocates into the nucleus where, together with other transcription factors such as CSL and MAM, it stimulates the transcription of target genes such as HES (9). Notch-1 signaling plays a central role in the regulation of cell differentiation (10). However, Notch signaling is not restricted to development, and abnormal activation of Notch signaling has been implicated in the pathogenesis of human diseases such as T cell acute lymphoblastic leukemia and melanoma (11, 12). In this context, the first clinical trials with γ-secretase inhibitors have demonstrated promising results (13). Further clinical trials are currently analyzing inhibition of Notch signaling for the treatment of solid tumors such as gastrointestinal neuroendocrine tumors, metastatic thyroid cancer, and advanced breast cancer (www.clinicaltrials.gov).

In the present study we demonstrated that the Notch pathway is activated in the skin of patients with SSc. Inhibition of the Notch pathway with antisense constructs or with inhibitors of the γ-secretase complex prevented fibrosis in different animal models and induced the regression of established fibrosis. Thus, Notch-1 pathway inhibition might be a novel strategy for the treatment of SSc and other fibrotic diseases.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Patient and control skin biopsy samples.

Biopsy specimens were obtained from involved forearm skin of 11 patients with SSc (8 women and 3 men; median age 45 years [range 33–62 years]). All patients fulfilled the criteria for SSc described by LeRoy and Medsger (14); 6 had limited cutaneous SSc and 5 had diffuse cutaneous SSc. The median duration of SSc was 5 years (range 1–12 years). Control skin biopsy specimens were obtained from healthy age- and sex-matched volunteers (n = 8). All patients and controls signed consent forms approved by the local institutional review boards.

Immunohistochemistry analysis for NICD and α-smooth muscle actin (α-SMA).

To investigate whether the Notch pathway is activated in SSc, immunohistochemistry analysis for the NICD was performed. Fresh frozen sections from SSc patients, healthy subjects, and patients with hypertrophic scars and keloids were incubated with 3% H2O2 followed by serum blocking with 10% goat serum in 5% bovine serum albumin (BSA). The NICD was detected by staining with polyclonal rabbit anti-human NICD antibodies (Abcam) overnight at 4°C.

Expression of α-SMA was analyzed in paraffin-embedded specimens obtained from mice (see below). After deparaffinization, samples were incubated with 3% BSA and then with 3% H2O2. Positivity for α-SMA in mouse sections was detected by incubation with anti–α-SMA monoclonal antibodies (clone 1A4; Sigma-Aldrich). Irrelevant isotype antibodies (Santa Cruz Biotechnology) in the same concentration were used as control, and antibodies labeled with horseradish peroxidase (Dako) were used as secondary antibodies. Expression of the NICD and α-SMA was visualized with diaminobenzidine peroxidase substrate solution (Sigma-Aldrich). The number of myofibroblasts was counted in 6 different sections of lesional skin from each mouse by an examiner who was blinded with regard to the treatment regimen.

Quantitative real-time polymerase chain reaction (PCR).

Total RNA was isolated with the NucleoSpin RNA II extraction system (Machery-Nagel) and converted into complementary DNA (cDNA) as previously described (15). The following primer pairs were used for SYBR Green real-time PCR: human HES-1 forward 5′-TACCCAGCCAGTGTCAAC-3′, reverse 5′-CAGATGCTGTCTTTGGTTTATCC-3′; murine HES-1 forward 5′-CCAAGCTAGAGAAGGCAGACA-3′, reverse 5′-CCGGAGGTGCTTCACAGT-3′. Quantification of β-actin was used to normalize for the amounts of loaded cDNA.

MTT assay.

The metabolic activity of dermal fibroblasts incubated with DAPT was measured using the MTT method as previously described (16). Untreated fibroblasts were used as negative controls, and all other results were normalized to those in untreated cells. Fibroblasts incubated with 50% DMSO were used as positive controls.

Prevention of bleomycin-induced experimental fibrosis.

Skin fibrosis was induced in pathogen-free 6-week-old female DBA/2 mice (Charles River) by local injection of bleomycin for 21 days (15). One hundred microliters of bleomycin dissolved in 0.9% NaCl at a concentration of 0.5 mg/ml was administered every other day by subcutaneous injection into defined areas of the upper back (n = 10 mice). As a control, mice (n = 10) were injected subcutaneously with 100 μl of 0.9% NaCl, the solvent for bleomycin. To investigate whether the γ-secretase inhibitor DAPT exerts antifibrotic effects in vivo, 2 groups of mice were treated with DAPT dissolved in 10% DMSO in 0.9% NaCl, administered by oral gavage at 1.5 mg/kg/day or 6.0 mg/kg/day (n = 7 in each group). These dosages are pharmacologically relevant and were used in previous studies (17, 18). After 21 days, the animals were killed by cervical dislocation.

The mouse model of bleomycin-induced dermal fibrosis was also used to evaluate outcome in Notch-1 antisense–transgenic mice upon challenge with bleomycin. Notch-1 antisense–transgenic mice have been previously described (17, 19). These mice appear phenotypically grossly normal, but protein levels of Notch-1 are reduced by 50% in several organs including the skin (17, 19). Genotyping of the mice was performed by PCR with Notch-1 primers (forward 5′-GCTCCCATTCATCAGTTC-3′, reverse 5′-TCTGTGAGAGTGAGCAGG-3′). For our experiments, Notch-1 antisense–transgenic mice were backcrossed onto a C57BL/6 background for at least 12 generations. Four groups of mice were analyzed: two groups consisted of transgenic mice (n = 7 in each group), and the other 2 groups consisted of wild-type littermates (n = 8 for each group). Dermal fibrosis was induced in 1 group of transgenic mice and 1 group of wild-type mice by repeated injection of bleomycin; the other 2 groups received sham treatment with subcutaneous injections of NaCl and served as controls.

All mouse experiments were approved by the local ethics committee.

Inhibition of Notch signaling in tight skin 1 mice.

In addition to the mouse model of bleomycin-induced dermal fibrosis, the tight skin mouse model of SSc was used to evaluate the antifibrotic potential of Notch signaling inhibition. Due to a dominant mutation in fibrillin 1, the phenotype of TSK-1 is characterized by increased hypodermal thickness (20). Four groups of mice were analyzed. The first group of TSK-1 mice (n = 7) was killed at the age of 5 weeks to evaluate the degree of fibrosis before antifibrotic treatment. Another group of TSK-1 mice (n = 5) was treated with DAPT at a concentration of 6 mg/kg/day by oral gavage, and a third TSK-1 group (n = 12) received the solvent DMSO. The last group (n = 21) consisted of pa/pa (control) mice, which also received sham treatment with the solvent DMSO. The treatment was started when the mice were 5 weeks old. After 5 weeks of treatment, the mice were killed by cervical dislocation.

In addition to treatment with the γ-secretase inhibitor DAPT, TSK-1 mice were crossed with Notch-1 antisense–transgenic mice to generate TSK-1 mice expressing the Notch as construct. The offspring, consisting of Notch-1 antisense–transgenic/TSK-1 mice (n = 5), wild-type TSK-1 mice (n = 8), Notch-1 antisense–transgenic mice not carrying the mutated TSK-1 allele (Notch-1 antisense–transgenic/pa mice) (n = 5), and wild-type/pa mice (n = 8) were killed at the age of 10 weeks to analyze hypodermal thickness, collagen content, and number of myofibroblasts.

Genotyping of TSK-1 mice was performed by PCR with mutated fibrillin 1/TSK-1 primers (forward 5′-GTTGGCAACTATACCTGCAT-3′, reverse 5′-CCTTTCCTGGTAACATAGGA-3′).

Treatment of established fibrosis.

Potential regression of established fibrosis with DAPT treatment was evaluated in a modified bleomycin model (21). Six-week-old DBA/2 mice were divided into 6 groups (n = 8 per group). The first group of mice was killed after 3 weeks of bleomycin challenge in order to analyze fibrotic changes before antifibrotic treatment. Another group of mice was killed after 6 weeks of bleomycin injections. The third group was injected for 3 weeks with bleomycin and afterwards for another 3 weeks with 0.9% NaCl to control for spontaneous regression of fibrosis. To analyze the potential of DAPT for treatment of established fibrosis, one group of mice challenged for 6 weeks with bleomycin received antifibrotic treatment with DAPT at a dosage of 6 mg/kg/day for the last 3 weeks only. The last 2 groups received subcutaneous injections of 0.9% NaCl for 3 weeks and 6 weeks, respectively.

Histologic analysis.

The injected skin areas were fixed in 4% formalin and embedded in paraffin. Histologic sections were stained with hematoxylin and eosin for assessment of dermal thickness at the injection sites. Dermal thickness was determined by measuring the largest distance between the epidermal–dermal junction and the dermal–subcutaneous fat junction as previously described (22), using a Nikon Eclipse 80i microscope at 200× magnification. The measurements were performed by an examiner who was blinded with regard to the treatment group.

Hydroxyproline assay.

To analyze the collagen content in skin samples, hydroxyproline assay was performed as previously described (23). After digestion of punch biopsy specimens (3 mm) in 6M HCl for 3 hours at 120°C, chloramine T (0.06M) was added and samples were mixed and incubated for 20 minutes at room temperature. Perchloric acid (3.15M) and 20% p-dimethylaminobenzaldehyde were added, and samples were incubated for an additional 20 minutes at 60°C. Absorbance at 557 nm was determined with a Spectra Max 190 microplate spectrophotometer (Molecular Devices).

Statistical analysis.

Data are expressed as the mean ± SEM. Wilcoxon's signed rank test for related samples and the Mann-Whitney U test for nonrelated samples were used for statistical analyses. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Activation of Notch signaling in SSc patients.

First we analyzed whether Notch signaling is activated in SSc. Using antibodies that selectively detect the NICD but not uncleaved, inactive Notch-1, we demonstrated activation of the Notch pathway in SSc patients. The NICD was barely detectable by immunohistochemistry in the skin of healthy individuals (Figure 1A). In contrast, all SSc patients were positive for the NICD. Intense staining of the NICD was particularly observed in fibroblasts (Figure 1A). In addition, levels of messenger RNA (mRNA) for the Notch target gene HES-1 were elevated by a mean ± SEM of 453 ± 30% in skin biopsy specimens from SSc patients as compared to those from healthy individuals (P < 0.05) (Figure 1B). Thus, Notch signaling is prominently activated in SSc patients, but not in healthy subjects.

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Figure 1. Notch signaling is activated in systemic sclerosis (SSc). A, The Notch intracellular domain (NICD), as a marker of active Notch signaling, was barely detectable by immunohistochemistry analysis of skin specimens of healthy controls. However, intense staining for the NICD was detected in skin specimens from SSc patients, particularly in fibroblasts. Representative results are shown. Original magnification × 200 for normal and SSc skin sections and isotype control; original magnification × 1,000 for fibroblasts. B, Levels of mRNA for the Notch target gene hes-1 were elevated in the skin of SSc patients compared to healthy controls. Values are the mean ± SEM. ∗ indicates statistically significant difference.

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We additionally analyzed the activation of Notch signaling in other fibrotic diseases and performed immunohistochemistry analysis for NICD in hypertrophic scars and keloids. We also observed an accumulation of the NICD in patients with hypertrophic scars and keloids. Similar to the findings in SSc, staining was prominent in fibroblasts, but also occurred in keratinocytes and endothelial cells (histologic images available from the corresponding author upon request).

Inhibition of Notch signaling prevents bleomycin-induced dermal fibrosis.

To investigate the role of the Notch pathway in fibrosis, we used bleomycin-induced dermal fibrosis in the mouse as a model for the early inflammatory stages of SSc (24). Increased activation of the Notch pathway with prominent staining for the NICD was observed in bleomycin-challenged mice compared to vehicle (NaCl)–injected controls. In particular, fibroblasts from bleomycin-challenged mice stained positively for NICD. Consistent with the increased NICD levels, transcription of the Notch target gene HES-1 was activated in the skin of bleomycin-injected mice. Levels of HES-1 were increased by a mean ± SEM of 323 ± 35% in bleomycin-challenged mice compared to controls (P = 0.02). This induction was completely prevented by treatment with DAPT, even at a low dosage of 1.5 mg/kg/day (histologic images and data available from the corresponding author upon request).

Next we investigated whether inhibition of the Notch pathway with DAPT can prevent bleomycin-induced fibrosis. We first performed MTT assays to assess whether the γ-secretase inhibitor DAPT affects cell proliferation. No changes in metabolic activity were observed with DAPT at either concentration tested (1 μM and 10 μM) (data not shown). Further, we incubated dermal fibroblasts to evaluate the effect of DAPT treatment on collagen synthesis. Incubation of fibroblasts with DAPT effectively reduced collagen release from SSc fibroblasts, but not from healthy controls (P < 0.05) (data available from the corresponding author upon request). Treatment of mice with DAPT was well tolerated, and no differences in body weight or other obvious signs of toxicity were observed between DAPT- and vehicle-treated groups. In bleomycin-injected mice without antifibrotic treatment, massive accumulation of collagen bundles with prominent dermal thickening was observed (Figure 2A). Treatment with DAPT reduced dermal thickening in a dose-dependent manner (Figure 2B). At 1.5 mg/kg/day, DAPT inhibited dermal thickening by a mean ± SEM of 58 ± 10% (P = 0.004 versus bleomycin treatment without DAPT). The increase in dermal thickness was completely prevented with DAPT at 6 mg/kg/day (P = 0.002) (Figure 2B).

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Figure 2. Inhibition of γ-secretase complex by DAPT prevents bleomycin-induced dermal fibrosis. A, Treatment with DAPT prevented fibrosis in bleomycin-challenged mice. Mice were injected subcutaneously with NaCl (control), with bleomycin alone, with bleomycin and DAPT at a dosage of 1.5 mg/kg/day (low), or with bleomycin and DAPT at a dosage of 6 mg/kg/day (high). Representative results are shown. Original magnification × 100. B–D, Treatment with DAPT at 1.5 mg/kg/day and 6 mg/kg/day prevented dermal thickening upon bleomycin challenge (B), caused a dose-dependent reduction of the collagen content in lesional skin as analyzed by hydroxyproline assay (C), and prevented differentiation of resting fibroblasts into myofibroblasts (D). Values are the mean ± SEM. ∗ indicates statistically significant difference.

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Consistent with the reduced dermal thickening, accumulation of lesional collagen and differentiation of myofibroblasts were also prevented by treatment with DAPT. DAPT reduced hydroxyproline content by 87 ± 6% compared to that observed with bleomycin treatment alone (P = 0.002) (Figure 2C). Treatment with DAPT also decreased myofibroblast counts in a dose-dependent manner, with almost complete inhibition at a dosage of 6 mg/kg/day (P = 0.001) (Figure 2D; histologic results available from the corresponding author upon request).

To further evaluate the mechanism by which inhibition of Notch signaling reduces fibrosis, we measured the expression of various cytokines in affected skin. As expected, levels of mRNA for interleukin-4 (IL-4), IL-6, transforming growth factor β (TGFβ), connective tissue growth factor (CTGF), and interferon-γ (IFNγ) were up-regulated in bleomycin-challenged mice. This up-regulation was completely prevented upon inhibition of Notch signaling by DAPT (P < 0.05 for all cytokines except for IFNγ); detailed data available from the corresponding author upon request.

To use a genetic approach to confirm the results obtained with the chemical inhibitor DAPT, we evaluated outcome in Notch-1 antisense–transgenic mice challenged with bleomycin. Notch-1 antisense–transgenic mice have significantly reduced Notch-1 signaling in several organs, including the skin (17, 19). No histologic differences in the skin were observed between wild-type and Notch-1 antisense–transgenic mice injected with NaCl (Figure 3A). However, in Notch-1 antisense–transgenic mice, bleomycin did not induce dermal fibrosis (Figure 3A). Dermal thickening upon bleomycin challenge was reduced by 85 ± 19% in Notch-1 antisense–transgenic mice (P = 0.003 versus bleomycin-injected wild-type mice) (Figure 3B). Collagen content in lesional skin was also significantly decreased (by 77 ± 7%; P = 0.002) (Figure 3C), and myofibroblast differentiation was almost completely prevented (P = 0.006) (Figure 3D).

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Figure 3. Notch-1 antisense–transgenic (as tg) mice are protected against bleomycin-induced fibrosis. A, Dermal fibrosis with accumulation of thickened collagen bundles was strongly reduced by knockdown of Notch-1. Wild-type and Notch-1 antisense–transgenic mice were injected with NaCl or challenged with bleomycin. Representative results are shown. Original magnification × 100. B–D, Overexpression of a Notch-1 antisense construct prevented dermal thickening upon bleomycin challenge (B), reduced collagen content in lesional skin as analyzed by hydroxyproline assay (C), and prevented differentiation of resting fibroblasts into myofibroblasts (D). Values are the mean ± SEM. ∗ indicates statistically significant difference.

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Inhibition of the Notch pathway corrects the tight skin phenotype.

We further analyzed the antifibrotic potential of DAPT in the TSK-1 mouse model, which resembles later stages of SSc characterized by endogenous activation of fibroblasts without inflammatory infiltration. Notch signaling was activated in TSK-1 mice, with increased staining for the NICD, particularly in fibroblasts. In addition to elevated NICD levels, expression of HES-1 was up-regulated in TSK-1 mice (by a mean ± SEM of 543 ± 31% compared to control mice not carrying the TSK-1 allele; P < 0.05). Treatment with DAPT completely reduced the expression of HES-1 to control levels (P < 0.05) (histologic images and data available from the corresponding author upon request).

Skin fibrosis in TSK-1 mice was already manifest at the age of 5 weeks, and the degree of fibrosis at this age was only slightly lower than that observed in 10-week-old TSK-1 mice (Figure 4). Treatment with the γ-secretase inhibitor DAPT ameliorated the histologic changes in these mice (Figure 4A). The prominent hypodermal thickening in TSK-1 mice was significantly decreased (by a mean ± SEM of 75 ± 4%) upon treatment with 6 mg/kg/day DAPT (P = 0.003) (Figure 4B). Moreover, the hydroxyproline content and the number of myofibroblasts were strongly reduced (90 ± 21%; P = 0.02 and 87 ± 28%; P = 0.003, respectively) (Figures 4C and D). Of note, hypodermal thickness and myofibroblast counts in mice treated with DAPT from the age of 5 weeks to the age of 10 weeks were not only reduced compared to those in 10-week-old mice, but were also significantly lower than those in 5-week-old TSK-1 mice, and thus below pretreatment levels (Figure 4). These findings show that treatment with DAPT not only prevents progression of fibrosis, but also induces regression of fibrosis in TSK-1 mice.

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Figure 4. Inhibition of the γ-secretase complex ameliorates the TSK-1 phenotype. A, Untreated TSK-1 mice were studied at ages 5 weeks and 10 weeks, and another group of TSK-1 mice was studied after 5 weeks of treatment with DAPT; pa/pa mice were studied as controls. Bars indicate hypodermal thickness. Representative results are shown. Original magnification × 40. B–D, Treatment of TSK-1 mice with DAPT reduced hypodermal thickening (B), collagen content in lesional skin as analyzed by hydroxyproline assay (C), and myofibroblast counts (D). Values are the mean ± SEM. ∗ and # indicate statistically significant difference (versus untreated 10-week-old and untreated 5-week-old TSK-1 mice, respectively).

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Finally, Notch-1 antisense–transgenic mice were interbred with TSK-1 mice to yield animals bearing the Notch-1 antisense construct and the TSK-1 allele. Collagen accumulation and tissue fibrosis were significantly reduced in Notch-1 antisense–transgenic/TSK-1 mice compared to wild-type/TSK-1 littermates (Figure 5A). Hypodermal thickening was significantly decreased in Notch-1 antisense–transgenic/TSK-1 mice (51 ± 11%; P = 0.004) (Figure 5B). Correspondingly, collagen content and the number of α-SMA–positive myofibroblasts were reduced, by 96 ± 5% and 79 ± 16%, respectively (P = 0.01 and P = 0.003, respectively) (Figures 5C and D).

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Figure 5. Overexpression of a Notch-1 antisense construct corrects histologic changes in TSK-1 mice. A, Tissue sections from wild-type mice without the TSK-1 allele (wild-type/pa), Notch-1 antisense–transgenic (as tg)/pa mice, Notch-1 wild-type/TSK-1 mice, and Notch-1 antisense–transgenic/TSK-1 mice were analyzed. Bars indicate hypodermal thickness. Representative results are shown. Original magnification × 40. B–D, Hypodermal thickening (B), collagen content in lesional skin as analyzed by hydroxyproline assay (C), and myofibroblast counts (D) were strongly reduced in TSK-1 mice bearing the Notch-1 antisense construct. Values are the mean ± SEM. ∗ indicates statistically significant difference.

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Regression of existing fibrosis with inhibition of Notch signaling.

In addition to its preventive effects, we investigated whether inhibition of Notch signaling could induce regression of fibrosis. Therefore, we evaluated the effects of DAPT in a modified bleomycin model of established fibrosis. The dermal thickening observed after 3 weeks of bleomycin challenge further progressed in mice challenged with bleomycin for 6 weeks (Figure 6A). However, dermal thickening in mice challenged with bleomycin for 6 weeks and treated with DAPT for the last 3 weeks was significantly less pronounced than that in mice treated with bleomycin alone for 6 weeks (P = 0.007). Moreover, the dermal thickness in the DAPT-treated group was also reduced compared to that in mice treated with bleomycin for 3 weeks followed by injections with vehicle for another 3 weeks (P = 0.007) (Figure 6B). Thus, treatment with DAPT not only prevented progression of fibrosis, but induced significant regression of fibrosis.

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Figure 6. Inhibition of Notch signaling induces regression of established fibrosis. A, Mice were treated with NaCl alone for 3 weeks or 6 weeks, with bleomycin alone for 3 weeks or 6 weeks, with bleomycin for 3 weeks followed by NaCl for 3 weeks, or with bleomycin for 6 weeks along with DAPT for the last 3 weeks. Progressive dermal thickening was observed with bleomycin challenge over time, but treatment with DAPT induced potent regression of established fibrosis. Representative results are shown. Original magnification × 100. B–D, Treatment with DAPT at 6 mg/kg/day during the last 3 weeks of bleomycin treatment reduced dermal thickness almost back to baseline levels (B), decreased collagen content in lesional skin as analyzed by hydroxyproline assay (C), and reduced myofibroblast counts to near normal (D). Values are the mean ± SEM. ∗ and # indicate statistically significant difference (versus mice injected with bleomycin for 3 weeks followed by injection with NaCl for another 3 weeks and versus mice treated with bleomycin alone for 6 weeks, respectively).

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Consistent with the above findings, a significant reduction in hydroxyproline content was observed in mice treated with DAPT during the last 3 weeks of bleomycin treatment (Figure 6C). Treatment with DAPT reduced the collagen content in lesional skin by 97 ± 9% compared to that in animals that received 3 weeks of bleomycin injections alone (P = 0.01) (Figure 6C). Similarly, differentiation of resting fibroblasts into myofibroblasts was effectively reduced (by 79 ± 12% compared to mice challenged with bleomycin for 3 weeks; P = 0.009) (Figure 6D).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The present findings demonstrate that Notch signaling is activated in SSc patients and in different models of experimental fibrosis, with accumulation of the NICD and increased transcription of the target gene HES-1. Particularly pronounced activation was observed in SSc fibroblasts. Inhibition of the Notch pathway either pharmacologically by blockade of the γ-secretase complex or by overexpression of a Notch antisense construct exerted potent antifibrotic effects. Inhibition of Notch signaling prevented the development of dermal fibrosis in inflammation-dependent models of dermal fibrosis such as bleomycin-induced fibrosis and in inflammation-independent fibrosis in TSK-1 mice. These data suggest that targeting of Notch directly reduces collagen synthesis in fibroblasts and might be effective in the early inflammatory stages of SSc as well as in later, noninflammatory stages, when the inflammatory infiltrates have already resolved. These results are consistent with those of a very recent study in which DAPT prevented hypochlorite-induced fibrosis (25).

We further showed that inhibition of Notch signaling by DAPT reduces the expression of profibrotic cytokines such as IL-4, IL-6, TGFβ, and CTGF. The profound decrease in IL-4 is consistent with the recent finding that IL-4 is a direct Notch target gene (26). In addition to prevention of fibrosis, targeting of Notch signaling potently induced regression of experimental fibrosis. Treatment with DAPT reduced dermal thickness, myofibroblast counts, and the hydroxyproline content of the skin almost back to levels observed in control mice.

We believe these results might have direct translational significance. The Notch pathway has been implicated in the pathogenesis of a variety of malignancies, and different strategies for inhibition of this pathway are currently under investigation for novel therapeutic approaches (13, 27). Studies analyzing the therapeutic potential of inhibitors of the γ-secretase complex that is required for release of the active NICD are currently the most common method for targeting Notch (13, 28). Two different inhibitors of the γ-secretase complex, MK0752 and PF-03084014, are currently being evaluated in clinical trials for the treatment of resistant T cell acute lymphoblastic leukemia and advanced breast cancer (www.clinicaltrials.gov). In addition, clinical trials of LY450139 in Alzheimer's disease are currently ongoing (www.clinicaltrials.gov).

However, targeting of γ-secretase is problematic, as all Notch receptors from Notch-1 to Notch-4 are affected. Thus, clinical trials with γ-secretase inhibitors have revealed several adverse events, such as gastrointestinal toxicity (29, 30). These side effects might be substantially ameliorated by brief interruptions in the administration of γ-secretase inhibitors. A short time period seems to be sufficient to allow at least some intestinal stem cells to correctly differentiate into enterocytes (28). To avoid these adverse events, other approaches to target the Notch pathway, such as use of antibodies specifically targeting individual Notch receptors or direct inhibition of the assembly of the transcription factor complex by synthetic, cell-permeable peptides, were recently evaluated and showed promising effects, without intestinal toxicity, in preclinical models (31, 32).

Thus, different pharmacologic inhibitors of the Notch pathway are available for clinical trials. Due to the prominent activation of the Notch pathway in experimental fibrosis and in human fibrotic skin, and the potent antifibrotic effects observed upon inhibition of Notch signaling at multiple experimental levels, targeting of Notch might be a promising novel therapeutic approach for the treatment of SSc. This is particularly important because the uncontrolled accumulation of extracellular matrix often leads to severe organ dysfunction with high morbidity and mortality, and because efficient antifibrotic therapies are not yet available (4, 6).

In summary, we have demonstrated that the Notch pathway is activated in SSc and that inhibition of Notch signaling exerts potent antifibrotic effects in preclinical models. Our data suggest that Notch signaling might be a promising molecular target for antifibrotic therapeutic approaches. Since inhibitors of the Notch pathway are currently being evaluated in clinical trials for other indications, these findings might lead to the initiation of clinical trials in patients with SSc and other fibrotic diseases.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. J. H. W. 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 conception and design. Dees, Akhmetshina, Zwerina, Mattson, O. Distler, Schett, J. H. W. Distler.

Acquisition of data. Dees, Zerr, Tomcik, Beyer, Horn, Palumbo, Reich, Sticherling.

Analysis and interpretation of data. Dees, Zerr, Tomcik, Zwerina, Mattson, O. Distler, J. H. W. Distler.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We would like to thank Maria Halter for excellent technical support.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES