Inhibition of Wnt/β-catenin/CREB binding protein (CBP) signaling reverses pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia is a ravaging condition of progressive lung scarring and destruction. Anti-inflammatory therapies including corticosteroids have limited efficacy in this ultimately fatal disorder. An important unmet need is to identify new agents that interact with key molecular pathways involved in the pathogenesis of pulmonary fibrosis to prevent progression or reverse fibrosis in these patients. Because aberrant activation of the Wnt/β-catenin signaling cascade occurs in lungs of patients with IPF, we have targeted this pathway for intervention in pulmonary fibrosis using ICG-001, a small molecule that specifically inhibits T-cell factor/β-catenin transcription in a cyclic AMP response-element binding protein binding protein (CBP)-dependent fashion. ICG-001 selectively blocks the β-catenin/CBP interaction without interfering with the β-catenin/p300 interaction. We report here that ICG-001 (5 mg/kg per day) significantly inhibits β-catenin signaling and attenuates bleomycin-induced lung fibrosis in mice, while concurrently preserving the epithelium. Administration of ICG-001 concurrent with bleomycin prevents fibrosis, and late administration is able to reverse established fibrosis and significantly improve survival. Because no effective treatment for IPF exists, selective inhibition of Wnt/β-catenin-dependent transcription suggests a potential unique therapeutic approach for pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia, the most common of the idiopathic interstitial pneumonias, is a devastating, progressive disorder characterized by excessive fibroblast proliferation and extracellular matrix remodeling leading to lung destruction. The incidence and prevalence of IPF in the United States appear to be increasing, with recent estimates of 6.8 to 16.3 cases per 100,000 and 14 to 42.7 cases per 100,000, respectively (1–4). There is currently no effective therapy for this uniformly fatal disease, which has a median survival of only 3 to 4 y.

Historically, inflammation has been viewed as central to the pathogenesis of IPF, with an initial inflammatory stimulus thought to initiate fibroblast activation and proliferation and progressive fibrosis. However, the lack of efficacy of anti-inflammatory agents has led to a re-evaluation of this concept (4). In the current paradigm, IPF is thought to be the result of injury to the alveolar epithelium that sets up a cascade of dysregulated epithelial-fibroblast crosstalk analogous to abnormal wound healing (5, 6). In this model, epithelial injury, through the release of growth factors (e.g., TGF-β1), cytokines, and matrix metalloproteinase (MMPs), leads to mesenchymal cell activation/proliferation, formation of fibroblastic foci, transformation of fibroblasts to myofibroblasts, and extracellular matrix deposition, culminating in parenchymal destruction (6). Myofibroblasts in turn induce apoptosis of epithelial cells, which together with basement membrane disruption, impedes epithelial repair. Consistent with a central role for the epithelium in disease pathogenesis, alveolar epithelial cells in IPF appear morphologically abnormal, with hyperplastic pneumocytes and reactive elongated cells [possibly intermediate between alveolar type II (AT2) and alveolar type I (AT1 cells)] overlying fibroblastic foci, the presumed sites of active fibrogenesis. A prerequisite for normal reepithelialization is that AT2 cells proliferate and subsequently differentiate into AT1 cells. Therefore, the presence of these intermediate phenotypes suggests that impaired transition of AT2 to AT1 cells contributes to abnormal epithelial repair. Recent studies also suggest that injured epithelial cells themselves may give rise to fibroblasts and myofibroblasts, through a process of epithelial-mesenchymal transition (EMT), thereby contributing to fibroblast accumulation (7, 8).

The Wnt/β-catenin pathway initiates a signaling cascade critical in normal development of multiple organ systems, including the brain, intestines, skin, and lung (9). The hallmark of this pathway is that it activates the transcriptional role of the multifunctional protein β-catenin. Canonical Wnt signaling inactivates GSK-3β, preventing β-catenin phosphorylation. This action leads to accumulation of hypophosphorylated β-catenin in the cytoplasm and subsequent translocation to the nucleus, where it regulates target gene expression through interactions with members of the T-cell factor (TCF)/lymphoid enhancer factor family of transcription factors (reviewed in ref. 10). To generate a transcriptionally active complex, β-catenin recruits the transcriptional coactivators, cyclic AMP response-element binding protein binding protein (CBP) or its closely related homolog, p300, as well as other components of the basal transcription machinery (10–12). Activated Wnt/β-catenin signaling has been implicated in fibrosis in a number of organs, including lung, suggesting that this developmental pathway can be reactivated in adult tissues following injury (13–16). In this regard, analysis of β-catenin expression in IPF lungs revealed an increase in the number of cells expressing nuclear (activated) β-catenin in association with up-regulation of Wnt target genes (16). It was suggested that up-regulation of Wnt signaling may promote proliferation of AT2 cells and inhibit their ability to differentiate into AT1 cells (9), impairing the repair process and, given the importance of Wnt/β-catenin to EMT in other settings, may also induce injured AT2 cells to undergo EMT. Consistent with these results, microarray analysis revealed up-regulation of genes related to the Wnt/β-catenin pathway (17), and in a more detailed analysis that included primary AT2 cells derived from IPF patients, components of the Wnt signaling pathway and its downstream targets were increased at both gene and protein levels (16).

Wnt/β-catenin pathway activation has been shown to either promote or inhibit differentiation depending on the experimental circumstances, without a clear understanding of the underlying mechanisms. We recently identified ICG-001, a unique small molecule that selectively inhibits TCF/β-catenin transcription in a CBP-dependent fashion, resulting in down-regulation of a subset of target genes [e.g., S100A4/fibroblast-specific protein-1 (FSP-1), cyclin D1, and survivin] that have been implicated in fibrosis (18–20). ICG-001 selectively blocks the β-catenin/CBP interaction without interfering with the β-catenin/p300 interaction (18, 20). Although p300 and CBP have to date generally been regarded as interchangeable with regard to downstream effects, our studies with ICG-001 suggest that differential effects of Wnt signaling on target genes may in fact be determined by differential coactivator usage (18, 21). Based on our studies with ICG-001, we proposed a model in which β-catenin/CBP-driven transcription is critical for maintenance of an undifferentiated/proliferative state (e.g., in both normal and cancer stem/progenitor cells, fibroblast proliferation), whereas a switch to β-catenin/p300-mediated gene expression is an essential first step in initiating normal cellular differentiation (19). Given that a number of CBP-dependent target genes are up-regulated in IPF (16), aberrant regulation of the balance between these two distinct but related β-catenin-dependent transcriptional programs may be important in the development of pulmonary fibrosis.

Using a well-established murine model of pulmonary fibrosis (22), we demonstrate that β-catenin signaling is activated following administration of bleomycin and abrogated by ICG-001. Furthermore, ICG-001, which specifically inhibits Wnt/β-catenin/CBP-driven transcription, not only prevents but reverses established fibrosis, demonstrating a causal role for aberrant Wnt signaling in lung fibrosis and suggesting a potential previously unexplored therapeutic approach for IPF.

Results

ICG-001 Inhibits Activation of Wnt/β-Catenin Signaling in the Lung and Prevents Bleomycin-Induced Pulmonary Fibrosis.

We first examined the effect of ICG-001 on activation of Wnt/β-catenin signaling induced by bleomycin in vivo as assessed by β-galactosidase activity in the β-catenin-activated transgene driving expression of nuclear β-galactosidase (BAT-gal) transgenic mice (23). BAT-gal mice, reliable reporters of Wnt/β-catenin activity (23), had marked lacZ expression as detected by X-gal staining in both alveolar (Fig. 1A) and airway epithelial cells after bleomycin treatment compared with saline controls (Fig. 1C). ICG-001 greatly decreased bleomycin-induced lacZ expression in the epithelium (Fig. 1B) consistent with an overall inhibitory effect on Wnt/β-catenin signaling in this model of pulmonary fibrosis. Quantitative real-time PCR (qPCR) demonstrated that bleomycin significantly increased the pulmonary expression of a number of well-documented Wnt/β-catenin target genes as well as genes associated with EMT, ECM remodeling, and fibrosis [i.e., S100A4, cyclin D1, stromelysin-1 (MMP-3), matrilysin (MMP-7), fibronectin, connective tissue growth factor (CTGF), TGF-β1, and collagen genes COL1α2, COL3α1, and COL6α1] (Fig. 1D). The increased expression of these genes was abolished in bleomycin-treated mice given ICG-001 over a 10-d period starting at d 0 (Fig. 1D). Lung sections from BAT-gal mice on day 42 following administration of bleomycin were immunostained for α-smooth muscle actin (SMA). In saline controls (Fig. 1E, Left), α-SMA staining is restricted to the vessel walls. Administration of bleomycin resulted in increased α-SMA expression in the interstitium (Fig. 1E, Center) that was reduced by administration of ICG-001 from days 21 to 42 (Fig. 1E, Right). Furthermore, as judged by qPCR, administration of ICG-001 decreased α-SMA induction by bleomycin (Fig. 1F). Similarly, in the rat type II epithelial cell line, RLE-6TN, ICG-001 significantly decreased TGF-β1 induction of α-SMA and collagen Iα2 as determined by qPCR, consistent with inhibition by ICG-001 of EMT (Fig. 1G). We next determined the effect of ICG-001 on the Wnt/β-catenin-dependent gene S100A4/FSP-1 (18) in IPF patient lung fibroblasts. ICG-001 significantly decreased expression of the β-catenin/CBP-dependent gene S100A4 by qPCR (Fig. 1H). The calcium-binding protein S100A4, (i.e., FSP-1), has been suggested to be a fibroblast-specific marker and hallmark of EMT, which may represent a common pathway in diseases marked by airway remodeling (9, 24) and reduction of FSP-1 expression in fibroblasts may be associated with mesenchymal-epithelial transition. We conclude that bleomycin activates Wnt/β-catenin signaling in the lung, and that ICG-001 effectively decreases the bleomycin-induced aberrant Wnt/β-catenin signaling in both the epithelium and in fibroblasts.

Fig. 1.

ICG-001 blocks bleomycin-induced activation of Wnt/β-catenin signaling in bleomycin mouse model. (A–C) The lacZ expression as assessed by X-gal staining. (A) Marked lacZ expression (arrows) was seen on day 10 in alveolar epithelial cells of BAT-gal mice administered bleomycin on day 1. (B) X-gal staining (arrows) was markedly reduced in bleomycin-treated mice given Wnt/β-catenin inhibitor ICG-001 (5 mg/kg per day) from days 0 to 10, and (C) absent in saline controls. Each group, n = 10. (Scale bar, 50 μm.) (D) Quantitative PCR was performed using RNA extracted from bleomycin-treated mice given either saline or ICG-001 from days 21 to 42 after initial bleomycin injury. By qPCR, the significantly increased expression of S100A4, cyclin D1, MMP-3, MMP-7, fibronectin, CTGF, TGF-β1, COL1α2, COL3α1, and COL6α1 in lungs of bleomycin-treated mice administered saline (black bars) [versus saline controls (gray bars)] was inhibited by 5 mg/kg per day ICG-001 (blue bars) given days 0 to 10. Error bars indicate SEM (n = 3). *P < 0.05 versus Bleo/Saline group by ANOVA. (E) Immunofluorescence microscopy of representative lung sections from mice on day 42 following administration of bleomycin ± ICG-001. Alpha-SMA (green) is not present in the interstitium of saline controls (Left). Administration of bleomycin results in increased α-SMA expression (Center) that is reduced by ICG-001 treatment (Right). Nuclei are labeled with propidium iodide (PI, red). (F) qPCR was performed using RNA extracted from mouse lungs extracted from mouse lungs treated with ICG-001 from day 21 to 42 after bleomycin injury. ICG-001 decreased α-SMA 42.1 ± 3.1% (n = 4, P < 0.05, compared to bleo/saline). (G) RLE-6TN cells were treated with TGF-β1 (0.5 ng/mL) in the presence or absence of ICG-001 (10 μM) for 24 h and RNA obtained. By qPCR, the increased expression of α-SMA and collagen 1α induced by TGF-β1 compared to vehicle-treated controls was significantly reduced by ICG-001 treatment. *P < 0.05 versus the group treated with TGF-β1 alone by ANOVA. (H) Effect of ICG-001 (5 μM) on β-catenin/CBP-dependent S100A4/FSP-1 gene expression in lung fibroblasts from IPF patients as determined by qPCR.

We next investigated whether pretreatment with ICG-001 could prevent lung fibrosis induced by bleomycin. As seen by histopathology and morphometry, intranasal administration of 0.08 units bleomycin on day 1 induced a dense infiltration of inflammatory cells into the lung tissue and extensive deposition of collagen in the airway (Fig. 2A and Fig. S1B) and alveolar regions on day 10 (Fig. 2B) compared with saline controls (Fig. 2 E and F, and Fig. S1A). ICG-001 (5 mg/kg per day) administration, beginning 1 d before bleomycin dosing and continuing via minipump infusion for the 10-d period of investigation, significantly reduced both the influx of inflammatory cells around the airways (Fig. 2C and Fig. S1C) and alveoli (Fig. 2 D and G) and the severity of pulmonary fibrosis around the airways (Fig. 2C and Fig. S1C) and alveoli (Fig. 2D) as assessed by Sirius red and Masson's trichrome staining for collagen. ICG-001 also significantly reduced fibrosis in the lung parenchyma as quantified with the well-validated Ashcroft grading system [Fig. 2G (Bleo/001 versus Bleo/Saline)], using a predetermined 0 to 8 scale of severity (25). Although the bleomycin-induced inflammatory cell infiltrate was significantly reduced by 1 mg/kg per day dexamethasone administered from day 0 to 10 [Fig. 2G (Bleo/Dex versus Bleo/Saline)], lung fibrosis was unaffected by corticosteroid intervention [Fig. 2G (Bleo/Dex versus Bleo/Saline) and Fig. S1 D vs. B]. Of note, ICG-001 maintained the airway epithelial cell layer (Fig. S2B) that was severely disrupted by bleomycin (Fig. S2A) compared with mice administered saline alone (Fig. S2C).

Fig. 2.

ICG-001 prevents pulmonary fibrosis in bleomycin-treated mice. Compared with saline controls [E, F, and G (Saline)], an intense inflammatory cell infiltration and markedly increased collagen deposition (arrows) was seen surrounding the airways (A) and in the alveolar region [B, G (Bleo/Saline)] on day 10 in lungs of mice given bleomycin on day 1 and saline days 0 to 10 by Sirius red stain (A, B) and Ashcroft score for grading lung fibrosis [G (Bleo/Saline)]. Fibrosis was substantially reduced by 5 mg/kg per day ICG-001 administered from days 0 to 10 [C, D, and G (Bleo/001)] but not by 1 mg/kg per day dexamethasone [G (Bleo/Dex)]. Both ICG-001 and dexamethasone treatment significantly reduced the inflammatory cell infiltration of the alveoli [G (Bleo/001) and (Bleo/Dex)]. (Scale bars, 50 μm.) Error bars indicate SEM (n = 5–6). *P < 0.05 versus Bleo/Saline group by ANOVA.

ICG-001 Intervention Influences Survival and Is Effective in Established Pulmonary Fibrosis.

We first examined the relative efficacy on survival in the inflammatory and early fibrogenic response period in the mouse bleomycin model of ICG-001 versus the selective cytokine modulator pirfenidone that has a reported antifibrotic effect in this model (26, 27). Less than 40% of mice survived 15 d after bleomycin treatment in the control group treated with saline from days 5 to 15 (Fig. S3). Daily treatment beginning on day 5 with pirfenidone had no significant effect on bleomycin-induced mortality (Fig. S3). In stark contrast, no deaths occurred in the bleomycin-treated group given ICG-001 from days 5 to 15 (Fig. S3).

To evaluate the efficacy of inhibiting Wnt/β-catenin/CBP-dependent transcription in a more clinically relevant setting, we next examined the effect on fibrosis of delayed administration of ICG-001 after resolution of the early inflammatory response to bleomycin (27). In these longer-term studies, ICG-001 given at 5 mg/kg per day from day 21 to 42 after bleomycin administration, attenuated (Fig. 3 C and D) the persistent lung fibrosis observed by Masson's trichrome staining for collagen in bleomycin-treated mice (Fig. 3 A and B) compared with mice administered saline alone (Fig. 3 E and F) and as determined by Ashcroft score [Fig. 3G (Bleo/001 versus Bleo/Saline)] and hydroxyproline/collagen content [Fig. 3H (Bleo/001 versus Bleo/Saline)]. ICG-001 also had an important effect on sparing airway epithelial cells by reducing the extensive apoptosis observed in the alveoli of bleomycin-treated mice (Fig. S4). Strikingly, nine of nine mice administered ICG-001 from days 21 to 42 after bleomycin treatment [Fig. 3I (Bleo/001)] survived to day 42, in contrast with only four of nine (44%) bleomycin-treated mice given saline [Fig. 3I (Bleo/Saline); P = 0.029, Bleo/Saline versus Bleo/001]. We conclude that late administration of ICG-001 ameliorates and reverses bleomycin-induced pulmonary fibrosis by selectively blocking Wnt/β-catenin/CBP-dependent signaling.

Fig. 3.

ICG-001 reverses pulmonary fibrosis and protects against lethality induced by bleomycin. The effect of ICG-001 on established persistent pulmonary fibrosis (A–H) and survivability (I) in the period after resolution of the early inflammatory response to bleomycin was determined (n = 9 per group). In mice given bleomycin on day 0 and administered saline from days 21 to 42, extensive lung fibrosis as assessed by Masson's trichrome stain (A and B), Ashcroft score [G (Bleo/Saline)], and increased hydroxyproline/collagen content [H (Bleo/Saline)] was observed compared with saline controls [E, F, and G, H (Saline)]. Treatment for the 21-d period with ICG-001 (5 mg/kg per day) reversed the pulmonary fibrosis [C, D, and G, H (Bleo/001)]. Error bars indicate SEM. *P < 0.05 versus Bleo/Saline group by ANOVA]. (I) ICG-001 (—●—) significantly reduced the mortality seen in bleomycin-treated mice given saline (—○—). *P = 0.029 by Fisher's exact test, Bleo/Saline versus Bleo/001.

Discussion

An emerging paradigm proposes a central role for alveolar epithelial cell injury and dysregulated repair in the pathogenesis of IPF. Injury to the epithelium is thought to initiate a cascade of fibroblast activation and matrix deposition, which in predisposed hosts fails to resolve as it would in the course of normal wound repair. Epithelial cells undergo excessive apoptosis, whereas fibroblasts are less amenable to apoptosis and manifest increased survival. The well-characterized model of bleomycin-induced lung injury has been extensively used to investigate potential pathways involved in the pathogenesis of pulmonary fibrosis and to explore therapeutic approaches (22). Despite some limitations with regard to recapitulation of human disease, a number of pathways that are up-regulated in IPF (e.g., TGF-β and Wnt/β-catenin) are also up-regulated following bleomycin (28). As reviewed in ref. 27, this model in rodents is characterized by a biphasic response, with an early inflammatory phase followed by a fibrotic phase that is evident by day 14 with maximal responses between days 21 and 28. Initial injury denudes the alveolar airspaces and exposes subepithelial basement membrane. There is evidence of aberrant alveolar epithelial repair, with increased metaplastic alveolar epithelial cells that apparently do not properly differentiate to a type I phenotype (29). Inflammatory cells are recruited to sites of epithelial injury, with subsequent release of cytokines and growth factors that promote matrix deposition and fibroblast, as well as epithelial proliferation (30). The increased fibroblasts in the lung, whether recruited from outside the lung (31) or generated via EMT (32), subsequently proliferate and deposit extensive extracellular matrix (e.g., fibronectin, collagen type I and III).

Using the bleomycin-induced model of pulmonary fibrosis in transgenic BAT-gal mice, we demonstrate in the current study that aberrant activation of Wnt signaling in the lungs is induced after insult. Intranasal administration of bleomycin caused marked lacZ expression in the airway and alveolar epithelium of BAT-gal transgenic mice, which was significantly reduced by the specific inhibitor of Wnt/β-catenin/CBP-driven transcription, ICG-001 (18–21). Bleomycin treatment also dramatically increased expression of a number of genes specifically associated with fibrosis, ECM deposition, and EMT (e.g., S100A4, MMP-7, CTGF, collagen types I and III, fibronectin, and TGF-β1) including several Wnt/β-catenin target genes (24). Treatment with ICG-001 reduced the expression of these genes essentially to the level of control.

Recent evidence suggests a significant role for EMT in fibrosis following administration of bleomycin treatment (33, 34). ICG-001 significantly decreased the expression of S100A4/FSP-1 both in the bleomycin-induced fibrosis model in vivo in the mouse and in fibroblasts from IPF patients in vitro. S100A4/FSP-1 has been suggested to be a hallmark of EMT that may represent a common pathway in diseases marked by airway remodeling (9). We associate the reduction in S100A4/FSP-1 expression via CBP/β-catenin antagonism to be associated with mesenchymal-epithelial transition. Furthermore, we demonstrated in vitro using rat type II lung epithelium that ICG-001 prevents the TGF-β1-induced up-regulation of α-SMA and type I collagen, genes that are typically increased in EMT. ICG-001 pretreatment significantly reduced the severity of pulmonary fibrosis around airways and alveoli collagen. In marked contrast, although administration of dexamethasone (1 mg/kg per day) for the 10-d period significantly reduced the inflammatory cell infiltrate, interstitial and alveolar fibrosis were unaffected consistent with prior reports of failure of corticosteroids to ameliorate pulmonary fibrosis in either animal models or patients with lung fibroproliferative disorders (35). This observation is also consistent with a number of studies that have failed to demonstrate a prominent role for inflammation in the pathogenesis of human pulmonary fibrosis (reviewed in ref. 36). To examine the critical therapeutic question of whether established airway and interstitial fibrosis can be reversed, we examined the effects of blockade of Wnt/β-catenin/CBP-mediated signaling during the later period of pulmonary fibrosis (27), after resolution of the early inflammatory response to bleomycin (27). In these longer-term studies, ICG-001 given from day 21 to 42 after bleomycin administration significantly reduced interstitial fibrosis and collagen deposition. Strikingly, nine of nine mice administered ICG-001 from days 21 to 42 after bleomycin treatment survived to day 42, in contrast with only four of nine (44%) of bleomycin-treated mice given saline. Pirfenidone, a selective cytokine modulator that was recently approved for the treatment of IPF in Japan, had no significant effect on bleomycin-induced mortality. Furthermore, ICG-001 maintained the airway epithelial cell layer (Fig. S2B) that was severely disrupted by bleomycin (Fig. S2A) compared with mice administered saline alone (Fig. S2C) and significantly decreased airway epithelial cell apoptosis (Fig. S4).

Wnt signaling plays an essential role in development and maintenance of multiple organ systems, including the brain, intestines, hematopoietic, skin, and lung (9, 10, 37). A number of Wnt genes, including Wnt2, Wnt5a, Wnt7b, Wnt11, and Wnt13 are expressed in both developing and adult lung (9). Recent studies also demonstrate activation of Wnt signaling in IPF, suggesting a role for this pathway in the pathogenesis of human lung fibrosis. In this regard, increased expression of two TCF/β-catenin-regulated genes (i.e., cyclin D1 and MMP-7) was observed in the airways of IPF patients (15, 16). Lungs of IPF patients demonstrate increased expression of several Wnt family members (e.g., Wnt1-inducible signal pathway protein and secreted frizzled related protein 1), although accumulation of nuclear β-catenin, a hallmark of activated Wnt signaling, has been observed in both epithelial (AT2) and mesenchymal (myofibroblasts) cells in lungs of IPF patients (15, 38, 39). A dichotomous role of Wnt signaling in stem cells, organ development, maintenance, and repair is a commonly observed phenomenon (40), with Wnt signaling playing roles in the seemingly opposing processes of cell proliferation and differentiation. Although a critical role for Wnt signaling in differentiation of the postnatal lung has been demonstrated (41), activation of Wnt signaling in IPF has been suggested to play a role in the increased proliferation of AT2 cells while preventing normal differentiation (9). We have recently developed a model that rationalizes these divergent responses to activation of Wnt/β-catenin signaling (18–21). The model posits that increased CBP/β-catenin-mediated transcription is associated with proliferation without differentiation, whereas a switch to the p300/β-catenin interaction is a requisite first step to initiate differentiation (19). We further propose that aberrant coactivator usage (i.e., increased CBP usage at the expense of p300) by β-catenin may be responsible for the improper termination of the wound healing process, maintaining epithelial cell proliferation and inhibiting differentiation. Aberrant regulation of the balance between these two related transcriptional programs may be important in the development, progression, and persistence of pulmonary fibrosis. Consistent with this finding, treatment with ICG-001, a selective inhibitor of Wnt/β-catenin-CBP-dependent transcription, decreased aberrant Wnt activation and Wnt target gene expression in the mouse model of bleomycin-induced pulmonary fibrosis, and not only halted disease progression but reversed established injury, enhanced repair, and dramatically decreased mortality.

An important unmet need is to identify new agents that effectively treat the devastating effects of fibrotic disorders to both prevent progression and reverse fibrosis in patients. Amelioration of fibrosis, together with improved survival following both concurrent and late administration of ICG-001, support an important role for the Wnt signaling pathway in the pathogenesis of fibrosis and provide strong rationale for selective blockade of Wnt/β-catenin-CBP-dependent transcription signaling as a potential previously unexplored therapeutic strategy for amelioration of human fibrotic lung disease.

Materials and Methods

Bleomycin-Induced Pulmonary Fibrosis in Mice.

All animal use procedures were approved by the University of Washington Animal Care Committee. In prevention of pulmonary fibrosis studies, C57BL/6J (The Jackson Laboratory) mice and BAT-gal transgenic mice that express the Escherichia coli lacZ gene encoding β-galactosidase under the control of β-catenin/TCF responsive elements produced from B6D2F1 mice (Charles River Breeding Laboratories) (23) had s.c. placement of miniosmotic pumps (200 μL Alzet Model 2001 osmotic pumps, 1.0 ± 0.15 μL/h delivery rate; Durect Corporation) containing ICG-001 (18) (5 mg/kg per day in saline, Institute for Chemical Genomics), dexamethasone-water soluble (1 mg/kg per day in saline; Sigma-Aldrich Corporation), or saline control on day 0. Twenty-four hours later (day 1), 0.08 units bleomycin (Novation LLC) (42, 43) in 50 μL of saline was administered intranasally. Control groups received 50 μL of saline intranasally on day 1. Mice were killed on day 10. In intervention studies during the inflammatory and early fibrogenic response period, C57BL/6J mice received bleomycin (0.08 units) or saline intranasally on day 0 followed on day 5 by twice daily oral treatment with pirfenidone [5-methyl-1-phenyl-2-(IH)-pyridone, 400 mg/kg/d in 0.5% carboxymethylcellulose; Sigma-Aldrich Corporation], or miniosmotic pump infusion of ICG-001 (5 mg/kg per day) or saline control for a 10-d treatment period. In long-term reversal of established fibrosis studies, groups of mice administered bleomycin (0.08 units intranasally) on day 0, had implantation on day 21 of miniosmotic pumps containing either ICG-001 (5 mg/kg per day) or saline, and killed on day 42. Lungs were obtained for histopathology, qPCR, and collagen assay.

Lung Fibrosis and Inflammation Assessment.

Lung tissue was embedded in paraffin and cut into 5-μm sections. The severity of pulmonary fibrosis was scored (0–8 scale) in lung sections stained for collagen with Sirius red or Masson's trichrome stains (43) by the histopathologist (E.Y.C.) blinded to the protocol design using the grading system described by Ashcroft et al. (25). The sections were stained with H&E to determine inflammatory cell infiltration on a semiquantitative scale ranging from 0 to 4 (44). Lung sections from BAT-gal transgenic mice underwent X-gal staining (23). Immunostaining for α-SMA was performed as previously described (7). Lung collagen was determined by hydroxyproline content (45).

Analysis of DNA Fragmentation in Lungs.

Immunohistochemical detection of apoptosis at the single-cell level in the lung tissue, based on labeling of DNA strand breaks (TUNEL technology), was performed using the In Situ Cell Death Detection Kit, POD (Roche Diagnostics GmbH) according to the manufacturer's instructions.

Treatment of Mouse Lung for qPCR.

Total lung RNA was isolated using an RNeasy mini kit (QIAGEN, Inc.), and mRNA levels for GAPDH determined by qPCR. A model 7900HT Fast Real-Time PCR System (Applied Biosystems) was used, and Applied Biosystems SYBR Green PCR master mix was used. RNA (0.2 μg) was used to synthesize first-strand cDNA with the SuperScript III First-Strand Synthesis System (Invitrogen Corporation). All primers were designed using Primer 3 and crossed large expanses of intronic sequence (Table S1). PCR sizes were ∼100 bp and confirmed by gel electrophoresis.

RLE-6TN Cell qPCR Studies.

To evaluate effects of ICG-001 on α-SMA and collagen type 1 expression, RLE-6TN cells were treated with TGF-β1 (0.25 ng/mL) in the presence or absence of ICG-001 (5.0 μM). After 24 h, cells were harvested and mRNA isolated for analysis by qPCR. RNA was reverse-transcribed using SuperScript reverse transcriptase (Invitrogen). Quantitative PCR was performed with SYBR-Green PCR using Real-Time PCR System HT7900 (Applied Biosystems). The amplification protocol was set as follows: 95 °C denaturation for 10 min followed by 40 cycles of 15-s denaturation at 95 °C, 1 min of annealing/extension, and data collection at 60 °C.

Primer pairs used are as following: α-SMA forward 5′-ATGGCTCCGGGCTCTGTAA-3′ and reverse 5′-ACAGCCCTGGGAGCATCA-3′; collagen 1α forward 5′-TTGACCCTAACCAAGGATGC-3′ and reverse 5′-CACCCCTTCTGCGTTGTATT-3′.

IPF Lung Fibroblasts.

Primary fibroblast cultures were derived from lung tissue from a patient with IPF undergoing transplant surgery, following informed consent and ethics approval from relevant institutions, as previously described (46). One cell line (CCL-134) established from a patient with IPF was obtained from ATCC. The IPF fibroblasts were treated with ICG-001 (5 μM) or DMSO control for 48 h, after which mRNA was isolated for analysis by qPCR.

Statistical Analysis.

The data are reported as the mean ± SEM. Differences were analyzed for significance (P < 0.05) by either ANOVA using the least-significant difference method or Fisher's exact test.