ORIGINAL RESEARCH Inhibition of b-Catenin Signaling Improves Alveolarization and Reduces Pulmonary Hypertension in Experimental Bronchopulmonary Dysplasia Deepthi Alapati, Min Rong, Shaoyi Chen, Dorothy Hehre, Stefanie C. Hummler, and Shu Wu Department of Pediatrics, Division of Neonatology, Batchelor Children’s Research Institute, University of Miami Miller School of Medicine, Miami, Florida
Abstract Bronchopulmonary dysplasia (BPD) is the most common and serious chronic lung disease of preterm infants. The development of pulmonary hypertension (PH) significantly increases the mortality and morbidity of this disease. b-Catenin signaling plays an important role in tissue development and remodeling. Aberrant b-catenin signaling is associated with clinical and experiment models of BPD. To test the hypothesis that inhibition of b-catenin signaling is beneficial in promoting alveolar and vascular development and preventing PH in experimental BPD, we examined the effects of ICG001, a newly developed pharmacological inhibitor of b-catenin, in preventing hyperoxia-induced BPD in neonatal rats. Newborn rat pups were randomized at postnatal day (P)2 to room air (RA) 1 DMSO (placebo), RA 1 ICG001, 90% FIO2 (O2) 1 DMSO, or O2 1 ICG001. ICG001 (10 mg/kg) or DMSO was given by daily intraperitoneal injection for 14 days during continuous exposure to RA or hyperoxia. Primary human pulmonary arterial smooth muscle cells (PASMCs) were cultured in RA or hyperoxia (95% O2) in the presence of DMSO or ICG001 for 24 to 72 hours. Treatment with ICG001 significantly increased alveolarization and reduced pulmonary vascular remodeling and PH during hyperoxia.
Bronchopulmonary dysplasia (BPD) is the most common and serious chronic lung disease of premature infants (1). Over the past four decades, the incidence of BPD has significantly increased as a result of the improved survival of extremely low-birthweight infants (1–3). There is no effective therapy for BPD due to its multifactorial
Furthermore, administering ICG001 decreased PASMC proliferation and expression of extracellular matrix remodeling molecules in vitro under hyperoxia. Finally, these structural, cellular, and molecular effects of ICG001 were associated with downregulation of multiple b-catenin target genes. These data indicate that b-catenin signaling mediates hyperoxia-induced alveolar impairment and PH in neonatal animals. Targeting b-catenin may provide a novel strategy to alleviate BPD in preterm infants. Keywords: b-catenin; hyperoxia; neonatal lung injury;
bronchopulmonary dysplasia; pulmonary hypertension
Clinical Relevance Inhibition of b-catenin signaling improves alveolarization and reduces pulmonary vascular remodeling and pulmonary hypertension in a neonatal rat model of bronchopulmonary dysplasia. These findings suggest that b-catenin signaling inhibitors may have the potential as novel therapeutics for bronchopulmonary dysplasia.
etiology and poorly understood disease processes. BPD is increasingly being recognized as resulting from interactive mechanisms involving arrested normal lung development and abnormal repair of injury to the immature lung caused by oxygen toxicity, mechanical ventilation, and infection (4, 5). Pulmonary
hypertension (PH) often complicates severe BPD, and this significantly contributes to the mortality and morbidity of preterm infants (6, 7). Studies to investigate the pathogenesis of severe BPD complicated by PH and to identify potential therapeutic targets are urgently needed.
( Received in original form August 1, 2013; accepted in final form January 21, 2014 ) This work was supported by an American Heart Association Grant-in-Aid (S.W.); a Micah Batchelor Award from the Batchelor Foundation (S.W.); by Project NewBorn, University of Miami (S.W.); by the Marta Marx Fund from the Scleroderma Foundation (S.W.); and by Research Award-Ikaria (D.A.). Correspondence and requests for reprints should be addressed to Shu Wu, M.D., Department of Pediatrics/Division of Neonatology, University of Miami Miller School of Medicine, PO Box 016960, Miami, FL 33101. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contenta at www.atsjournals.org Am J Respir Cell Mol Biol Vol 51, Iss 1, pp 104–113, Jul 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0346OC on January 31, 2014 Internet address: www.atsjournals.org
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ORIGINAL RESEARCH b-Catenin is a key regulatory protein with bidirectional capacity to tightly regulate nuclear transcription and to affect cell migration and adhesion by closely interacting with the cytoskeleton and adherens junction cadherins (8–11). A multiprotein “destruction complex” continually phosphorylates b-catenin, flagging it for degradation by the ubiquitin/ proteosome system. Inhibition of this phosphorylation by activation of Wnt and integrin/integrin-linked kinase (ILK) signaling allows b-catenin to accumulate in the cytoplasm and enter the nucleus to form a transcriptional complex localized at gene promoters (12–15). b-Catenin recruits the transcription coactivators, cyclic AMP response-element binding protein (CBP), or its closely related homolog p300 and other components of the basal transcription machinery (16–18). The transcription complex formed by b-catenin and its DNAbinding partners known as lymphocyte enhancer factor/T-cell factor (TCF) is able to promote chromatin remodeling and transcriptional initiation in a cell type–specific and context-specific manner, thus playing a key role in embryonic development, cell proliferation, differentiation, and survival. b-Catenin signaling is one of the important signaling pathways that regulates normal lung development and homeostasis (19, 20). Aberrant b-catenin signaling is associated with abnormal epithelial proliferation, fibroproliferative repair, and lung matrix remodeling in response to acute and chronic lung injuries (21–24). In fact, inhibition of b-catenin transcription activity by ICG001, a newly developed small molecule, ameliorated and reversed pulmonary fibrosis in a mouse model of bleomycin-induced lung injury (25, 26). We and others have previously demonstrated that b-catenin signaling is increased in the lungs of hyperoxia-exposed neonatal rats, a widely used experimental model of BPD (27, 28). A recent study demonstrated that increased b-catenin is present in the mesenchymal stromal cells isolated from infants developing BPD and in a-smooth muscle actin (a-SMA)-positive myofibroblasts in the lungs of infants dying of BPD (29). Together, these data suggest that aberrant b-catenin signaling may play an important role in the pathogenesis of BPD. In the current study, we tested the hypothesis that inhibition of b-catenin
signaling is beneficial in promoting alveolar and vascular development and preventing PH in a hyperoxia-induced severe BPD model in neonatal rats. We demonstrate that treatment with ICG001 improves alveolar and vascular development, decreases pulmonary vascular remodeling, and reduces PH during hyperoxia. In cultured human pulmonary artery smooth muscle cells (PASMCs), treatment with ICG001 decreases cell proliferation and inhibits expression of a-SMA, fibronectin, and connective tissue growth factor (CTGF) under hyperoxia exposure. Thus, targeting b-catenin signaling may provide a novel strategy for preventing and treating BPD in preterm infants.
Materials and Methods Detailed descriptions of the materials and methods are provided in the online supplement. Animal Models
The animal protocol was approved by the University of Miami Institutional Animal Care and Use Committee. Newborn Sprague Dawley rat pups were randomized at postnatal day (P)2 to room air (RA) 1 DMSO (placebo), RA 1 ICG001 (a specific pharmacological inhibitor of b-catenin), 90% FIO2 (O2) 1 DMSO, and O2 1 ICG001. ICG001 (10 mg/kg) or DMSO (99%, equal volume) was given by daily intraperitoneal injection for 14 days during continuous RA or hyperoxia exposure. Hemodynamic Measurements
On P15, right ventricular systolic pressure (RVSP) and right ventricle (RV) to left ventricle (LV) plus septum (LV 1 S) weight ratio (RV/LV 1 S) were determined as indices for PH (30). Briefly, rats were sedated, tracheotomized, and ventilated with a Mini-Vent (Harvard Apparatus, Holliston, MA). After thoracotomy, a 25-gauge needle fitted to a pressure transducer was inserted into the RV. RVSP was measured and continuously recorded on a Gould polygraph (model TA-400; Gould Instruments, Cleveland, OH). Immediately after RVSP measurements, hearts were dissected for RV free wall separation from LV1S for RV/LV 1 S weight ratio measurements.
Alapati, Rong, Chen, et al.: b-Catenin Signaling in Experimental BPD
Lung Histology and Morphometry
Lungs were infused with 4% paraformaldehyde via a tracheal catheter at 20 cm H2O of pressure for 5 minutes, fixed overnight, and paraffin embedded. Hematoxylin and eosin–stained tissue sections were used to measure mean linear intercept (MLI), septal density, and radial alveolar count as previously described (27, 30, 31). Immunofluorescence and Double Immunofluorescence Staining
Immunofluorescence and double immunofluorescence staining were performed as previously described (27, 30). Pulmonary Vascular Morphometry
Pulmonary vascular density was determined by the average number of vonWillebrand factor–stained vessels (, 50 mm in diameter) from 10 random images on each lung section (27, 30). Assessment of Pulmonary Vascular Remodeling
Immunofluorescence staining for a-SMA was performed, and 20 peripheral pulmonary vessels (, 50 mm in diameter) were assessed for medial wall thickness (MWT) (27, 30). Proliferation of vascular smooth muscle cells (VSMCs) was assessed by double immunofluorescence staining for a-SMA and Ki67, a proliferation marker on lung sections. Expression of fibronectin was determined by immunofluorescence staining and real-time quantitative RT-PCR (qRT-PCR). Western Blot Analysis
Total protein extraction and Western blot analysis were performed as previously described (27, 30). Real-Time qRT-PCR
Total RNA isolation and real-time qRT-PCR was performed as previously described (30). PASMC Culture and Treatment
Human PASMCs were cultured according to the manufacturer’s instruction (Lonza, Portsmouth, NH) and used between passages 2 through 6. Cells were cultured in 10% FCS containing media to 60 to 70% confluence and then cultured in 1% FCS containing media for 24 hours. The cells were treated with ICG001 (10 mM) or DMSO and exposed to RA or 95% O2 for 24 to 72 hours in a sealed plastic chamber as previously described (32). 105
ORIGINAL RESEARCH Immunofluorescence staining for Ki67 was performed in cells grown on chamber slides. Cell proliferation index was determined by the ratio of Ki67-positive nuclei to total nuclei. Proteins were isolated from cells grown in 6-well plates for subsequent Western blot analysis. Statistical Analysis
Data were expressed as means 6 SD. Comparison among the groups was performed by one-way ANOVA followed by Student-Newman Keuls test. A P value , 0.05 was considered significant.
Results ICG001 Inhibits Hyperoxia Activation of b-Catenin Signaling In Vivo
We first examined b-catenin signaling by assessing b-catenin localization. Hyperoxia exposure induced diffuse b-catenin nuclear translocation in lungs treated with placebo or ICG001 (Figure 1A). Double immunofluorescence colocalized some of the b-catenin–positive nuclei in surfactant protein C (SP-C)–expressing cells, suggesting that b-catenin undergoes nuclear translocation in alveolar type II epithelial cells (Figure 1A). There was no significant difference in the index of b-catenin nuclear translocation between the hyperoxia plus placebo and hyperoxia plus ICG001 groups (Figures 1A and 1B). We then assessed expression of Wntinduced signaling protein-1 (WISP-1), a well-characterized b-catenin target gene. Hyperoxia exposure significantly increased WISP-1 expression in the placebo-treated lungs, and this was decreased by administration of ICG001 during hyperoxia (Figure 1C). Inhibition of b-Catenin Signaling Improves Alveolar development
Because hyperoxia exposure is known to impair alveolarization, we evaluated the effect of ICG001 on alveolar development by histology and morphometry. On histological examination, the lungs from the hyperoxia plus placebo group displayed large and simplified alveoli with fewer secondary septa (Figure 2A). Treatment with ICG001 improved alveolarization in hyperoxiaexposed lungs, which now appeared similar to RA-exposed lungs (Figure 2A). Morphometric analysis demonstrated 106
Figure 1. ICG001 inhibits b-catenin signaling. (A) Immunofluorescence staining for b-catenin (red signal), pro-surfactant C (pro-SP-C) (green signal), and DAPI nuclear staining (blue signal) were performed on lung sections from neonatal rats exposed to room air (RA) plus DMSO (RA 1 DMSO), RA 1 ICG001, hyperoxia plus DMSO (O2 1 DMSO), and O2 1 ICG001. Pink signals indicate colocalization of b-catenin with nuclei. Arrow indicates SP-C–expressing cells. In hyperoxia-exposed lungs, some of the b-catenin–positive nuclei were colocalized in pro-SPC–expressing cells. The b-catenin nuclear translocation (NT) index (% of b-catenin positive nuclei/total nuclei) was determined (B). Hyperoxia exposure in the presence of DMSO increased the b-catenin NT index compared with RA groups, and this was not affected by administration of ICG001. ***P , 0.001 compared with RA groups; †††P , 0.001 compared with RA groups (n = 5 per group). (C) Western blot analysis demonstrated that hyperoxia exposure increased Wntinduced signaling protein-1 (WISP1) expression in the placebo lungs, and this was significantly decreased by ICG001 treatment. Lane 1: RA 1 DMSO; lane 2: O2 1 DMSO; lane 3: RA 1 ICG001; lane 4: O2 1 ICG001. *P , 0.05 compared with RA groups; #P , 0.05 compared with O2 1 DMSO (n = 5 per group).
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ORIGINAL RESEARCH we next evaluated the effect of ICG001 on RVSP and RV/LV1S weight ratio as indices of PH and right ventricular hypertrophy (RVH), respectively. Compared with RA groups, the rats in the hyperoxia plus placebo group had significantly increased RVSP (Figure 4A) and RVH (Figure 4B). Administration of ICG001 during hyperoxia significantly decreased RVSP (17.80 6 2.56 versus 25.09 6 3.15, P , 0.001; O2 1 ICG001 versus O2 1 DMSO) and RV/LV1S weight ratio (0.34 6 0.05 versus 0.58 6 0.10, P , 0.001; O2 1 ICG001 versus O2 1 DMSO) (Figures 4A and 4B). These findings indicate that inhibition of b-catenin signaling protects against hyperoxia-induced PH in neonatal rats. Inhibition of b-Catenin Signaling Decreases Pulmonary Vascular Remodeling
Figure 2. Inhibition of b-catenin improves alveolarization. (A) Lung histology. Hyperoxia exposure increased mean linear intercept (MLI) (B), decreased septal density (C), and decreased radial alveolar count (RAC) (D) in the presence of DMSO as compared with RA. Administration of ICG001 decreased MLI, increased septal density, and increased RAC during hyperoxia. **P , 0.01 and ***P , 0.001 compared with RA; #P , 0.05 and ###P , 0.001 compared with O2 1 DMSO; † P , 0.05 compared with RA groups (n = 5 per group).
a significant increase in MLI in the hyperoxia lungs treated with the placebo as compared with the RA plus placebo group (51.23 6 3.06 versus 39.35 6 0.68, P , 0.001; O2 1 DMSO versus RA 1 DMSO) (Figure 2B), signifying larger and fewer alveoli. In contrast, treatment with ICG001 during hyperoxia significantly decreased MLI as compared with the corresponding hyperoxia group that received placebo (44.26 6 0.75 versus 51.23 6 3.06, P , 0.05; O2 1 ICG001 versus O2 1 DMSO) (Figure 2B). Correlating with decreased MLI, administration of ICG001 significantly increased secondary septal density as compared with placebo during hyperoxia (Figure 2C). Radial alveolar count was also significantly increased by ICG001 in hyperoxia-exposed lungs (Figure 2D). Thus, inhibition of b-catenin
signaling improves alveolar development during hyperoxia. Inhibition of b-Catenin Signaling Improves Vascular Development
Because hyperoxia exposure can inhibit pulmonary vascular development, we assessed vascular density in these lungs. Compared with RA-exposed lungs, hyperoxia exposure significantly decreased vascular density in the presence of the placebo (Figures 3A and 3B). Treatment with ICG001 modestly increased vascular density during hyperoxia as compared with the hyperoxia plus placebo group (Figures 3A and 3B). Inhibition of b-Catenin Signaling by ICG001 Reduces PH
Our previous studies have shown that hyperoxia exposure induces PH. Therefore,
Alapati, Rong, Chen, et al.: b-Catenin Signaling in Experimental BPD
Given that ICG001 attenuated hyperoxiainduced PH, we sought to elucidate whether inhibition of b-catenin signaling decreases pulmonary vascular remodeling. The degree of muscularization of peripheral pulmonary arterioles (, 50 mm in diameter) was determined by measuring the MWT on a-SMA–stained lung sections. The lungs in the hyperoxia plus placebo group had a significant increase in MWT compared with the RA plus placebo group (0.46 6 0.05 versus 0.21 6 0.03, P , 0.001; O2 1 DMSO versus RA 1 DMSO) (Figures 5A and 5B). However, treatment with ICG001 during hyperoxia decreased MWT to near RA levels (0.26 6 0.03 versus 0.46 6 0.05, P , 0.001; O2 1 ICG001 versus O2 1 DMSO). These data indicate that inhibition of b-catenin signaling prevents hyperoxia-induced excessive muscularization of peripheral pulmonary vasculature. Because abnormally increased proliferation of PASMCs contributes partly to increased muscularization of peripheral pulmonary arterioles in response to chronic lung injury, we assessed proliferation of PASMCs in Ki67 and a-SMA doublestained lung tissue sections. There were increased Ki67-positive PASMCs in the medial wall of peripheral pulmonary arterioles in the hyperoxia- and placeboexposed lungs as compared with RA groups (Figures 5C and 5D). In contrast, treatment with ICG001 significantly decreased the proliferation index of PASMCs as compared with the hyperoxia plus placebo group (18.20 6 5.41 versus 30.40 6 8.93, 107
ORIGINAL RESEARCH among the study groups (data not shown). Thus, ICG001 exerts direct antiproliferative effects on PASMCs and may contribute to the decreased proliferation of PASMCs observed in vivo. Expression of a-SMA in cultured PASMCs was markedly increased by hyperoxia exposure (0.72 6 0.01 versus 0.37 6 0.05, P , 0.001; O2 1 DMSO versus RA 1 DMSO) (Figure 6C). However, treatment of hyperoxia-exposed PASMCs with ICG001 decreased a-SMA expression to RA levels (0.28 6 0.01 versus 0.72 6 0.01, P , 0.001; O2 1 ICG001 versus O2 1 DMSO) (Figure 6C). CTGF and fibronectin are b-catenin target genes that play an important role in ECM deposition and in vascular remodeling (33–36). We thus evaluated whether inhibition of b-catenin signaling would down-regulate CTGF and fibronectin expression in PASMCs. Treatment with ICG001 significantly decreased expression of fibronectin and CTGF under hyperoxia (Figures 6D and 6E). Taken together, these findings highlight that b-catenin signaling plays a critical role in PASMC proliferation, differentiation, and remodeling in vitro.
Discussion Figure 3. Inhibition of b-catenin improves vascular development. (A) Immunofluorescence staining for vonWillebrand factor (vWF) (red signal) and DAPI nuclear staining (blue signal) was performed. (B) Vascular density (VD) was determined by counting vWF-positive vessels (, 50 mm) on 10 random high-power-field (HPF) images from each lung section. Hyperoxia exposure in the presence of the placebo significantly decreased VD as compared with RA groups. Administration of ICG001 significantly increased VD in hyperoxia-exposed lungs; however, it was still less than the RA groups. ***P , 0.001 compared with RA groups; †††P , 0.001 compared with RA groups; #P , 0.05 compared with O2 1 DMSO (n = 5–6 per group).
P , 0.01; O2 1 ICG001 versus O2 1 DMSO). Thus, inhibition of b-catenin signaling antagonizes hyperoxia-induced PASMC proliferation in neonatal lungs. Fibronectin is a b-catenin target gene that plays an important role in vascular remodeling (33, 34). We therefore assessed fibronectin protein and gene expression. In hyperoxia- and placebo-exposed lungs, there was increased fibronectin protein expression in the peripheral pulmonary vessels as compared with the lungs exposed to RA. Administration of ICG001 decreased vascular fibronectin expression during hyperoxia (Figure 5E). RT-qPCR confirmed that fibronectin gene expression was increased by hyperoxia and decreased by ICG001 treatment (Figure 5F). 108
Effects of ICG001 on PASMC Proliferation and Expression of ECM Remodeling Molecules In Vitro
Having observed the protective effects of ICG001 on PH and pulmonary vascular remodeling, we sought to further determine the effects of ICG001 on PASMC proliferation and expression of ECM remodeling molecules in vitro. Hyperoxia exposure in the presence of the placebo decreased PASMC proliferation (Figure 6A). Treatment with ICG001 decreased PASMC proliferation under RA and hyperoxia (Figure 6A). We also assessed cell apoptosis by examining expression of cleaved caspase-3 and found that there was no significant difference
In the present study, we demonstrate that hyperoxia exposure activates b-catenin signaling in the lungs of neonatal rats. Treatment with ICG001 inhibits b-catenin signaling and improves alveolarization and pulmonary vascular development in these animals. Inhibition of b-catenin signaling also drastically reduces RVSP and RVH, indicating decreased PH. The improvement in PH is associated with decreased proliferation of PASMCs in the medial wall of peripheral pulmonary vessels and a concomitant decrease in the MWT. In cultured PASMCs, inhibition of b-catenin signaling decreases cell proliferation and differentiation and down-regulates expression of the genes responsible for ECM remodeling. Collectively, these data suggest that inhibition of b-catenin signaling may interfere with the progression of hyperoxiainduced PH and pulmonary vascular remodeling through a direct inhibitory effect on PASMCs. This study therefore highlights the importance of b-catenin signaling in the pathogenesis of PH in a hyperoxiainduced rodent model of severe BPD and provides a potential therapeutic target for alleviating PH in infants with severe BPD.
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Figure 4. Inhibition of b-catenin decreases pulmonary hypertension. (A) Right ventricular systolic pressure (RVSP) was significantly increased in the O2 1 DMSO lungs as compared with RA groups. ICG001 administration significantly decreased RVSP during hyperoxia. ***P , 0.001 compared with the RA groups; †††P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO (n = 6–11 per group). (B) Right ventricular hypertrophy (RVH) was determined by the weight ratio of right ventricle to left ventricle 1 septum. Hyperoxia-exposed animals in the presence of the placebo presented with increased RVH, and this was significantly decreased by ICG001 treatment. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO (n = 6–11 per group).
b-Catenin normally controls cell–cell adhesion through its binding with cadherin in adherens junctions and mediates the link between cadherin and the actin cytoskeleton (8–11). In addition, b-catenin acts as a transcriptional activator and regulates transcription of target genes responsible for cell proliferation, apoptosis, and differentiation (12, 13). b-Catenin nuclear translocation can be activated by Wnt signaling and by integrin/ILK signaling (12–15). Within the nucleus, b-catenin forms a transcription complex with its DNA-binding partners lymphocyte enhancer factor/TCF. In this complex, b-catenin serves as an obligate coactivator through its ability to recruit CBP to promote chromatin remodeling and transcription initiation/elongation (16–18). ICG001 interacts with CBP and specifically blocks the b-catenin/CBP interface and down-regulates b-catenin/TCF signaling (25, 26, 37–39). Previous studies have demonstrated the efficacy of ICG001 in treating bleomycin-induced lung fibrosis in adult mice (26) and in blocking epithelial–mesenchymal transition in vitro (40). Hyperoxia-induced lung injury in neonatal rats has been widely used as an experimental model of BPD for mechanistic studies and therapeutic exploration. We and others have previously shown that chronic hyperoxia exposure increases b-catenin nuclear translocation and further signaling in the lungs of neonatal rats (27, 28, 41). In the present study, we confirmed that exposure to 90% O2 for 14 days increased b-catenin nuclear translocation and target gene expression and showed for the first time that inhibition of b-catenin–driven
nuclear transcription by ICG001 prevents hyperoxia-induced alveolar simplification. This is consistent with our previous findings that prevention of hyperoxiainduced b-catenin nuclear translocation by a neutralizing monoclonal antibody for CTGF resulted in a significant attenuation of hyperoxia-induced alveolar injury in neonatal rats (27). CTGF has the ability to activate b-catenin signaling through binding to Wnt coreceptor LRP5/6 and cell surface integrins. Although we have also previously showed that inhibition of Wnt receptor LRP5/6 by Mesd does not improve alveolarization during hyperoxia (41), Dasqupta and colleagues have previously shown that treatment with rosiglitazone, an agonist of peroxisome proliferator-activated receptor-g, protects against hyperoxiainduced alveolar damage by decreasing TGF-b and Wnt/b-catenin signaling (28). It is plausible to suggest that disruption of alveolarization by hyperoxia could be independent of LRP5/6 and that multiple other signaling pathways, such as integrin/ ILK and peroxisome proliferator-activated receptor-g, may play a more prominent role in b-catenin activation and alveolar development under hyperoxia. Nevertheless, this study signifies the critical role of b-catenin signaling in the pathogenesis of neonatal hyperoxia– induced alveolar impairment. The novel finding of the present study is that inhibition of b-catenin signaling significantly attenuates hyperoxia-induced PH. The reduced PH we observed was associated with a dramatic decrease in pulmonary vascular remodeling and a modest improvement in vascular density. This is important because PH contributes
Alapati, Rong, Chen, et al.: b-Catenin Signaling in Experimental BPD
significantly to the mortality and morbidity of preterm infants with severe BPD (6, 7). Despite the widespread availability and use of pulmonary vasodilators such as nitric oxide, the mortality rate of infants with BPD complicated by PH remains as high as 50% (42). This high mortality rate is associated with the pulmonary vascular changes observed in these patients. These include increased vascular tone and reactivity, hypertensive vascular remodeling, and decreased vascular growth (43–45). BPD is a multifactorial disease, and oxygen toxicity is one of the important causes. Although intermittent hypoxia has been shown to directly contribute to PH, extensive animal studies have demonstrated that hyperoxia exposure independently contributes to the vascular changes observed in BPD (46, 47). Therapeutic agents that target distinct pathogenic mechanisms of pulmonary vascular remodeling and growth in severe BPD may provide additional relief on PH and decrease mortality. As such, recent clinical trials have reported better results in adult PH using therapeutic agents that reversed lung vascular remodeling rather than prolonged vasodilatation (48, 49). Thus, targeting b-catenin signaling to alleviate pulmonary vascular remodeling may be a promising new therapy for PH in severe BPD. The dynamic process of vascular remodeling involves numerous molecular signaling cascades governing VSMC migration, differentiation, proliferation, and fate. Studies that focused on elucidating the underlying pathogenetic mechanisms of atherosclerosis and graft rejection implicated the cadherin family of cell adhesion molecules as having an important role in VSMC proliferation (50–53). Quasnicka and colleagues demonstrated that growth factors such as PDGF and bFGF that modulate human aortic VSMC proliferation do so via the activation of b-catenin/TCF signaling (54). Wang and colleagues also demonstrated that after balloon injury there were increased b-catenin expression and vascular remodeling in rat carotid artery VSMCs (55). In the same study, the authors described a dual role of b-catenin/TCF signaling in inhibiting apoptosis and promoting proliferation in VSMCs. We therefore used PASMC cultures to elucidate the underlying mechanisms by which inhibition of b-catenin signaling reduces 109
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Figure 5. Effects of ICG001 on pulmonary vascular remodeling. (A) Immunofluorescence staining for a-SMA (green signal) and DAPI nuclear staining (blue signal). (B) Medial wall thickness (MWT) was assessed from 20 peripheral vessels (, 50 mm in diameter) on each lung section. Hyperoxia increased MWT in the presence of placebo as compared with RA groups. ICG001 administration significantly decreased MWT in hyperoxiaexposed lungs. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO (n = 5 per group). (C) Double immunofluorescence staining for a-SMA (green signal), Ki67 (red signal), and DAPI nuclear staining (blue signal). (D) The PASMC proliferative index was determined as the percentage of Ki67positivea-SMApositive/a-SMApositive cells in vessels that are , 50 mm in diameter. Hyperoxia increased the PASMC proliferative index when exposed to the placebo. Treatment with ICG001 modestly decreased the PASMC proliferative index during hyperoxia. ***P , 0.001 compared with the RA groups; ††P , 0.01 compared with RA groups; ##P , 0.01 compared with O2 1 DMSO (n = 5 per group). (E) Immunofluorescence staining for fibronectin (green signal) and DAPI nuclear staining (blue signal). Fibronectin staining was increased in O2 1 DMSO lungs, and this was decreased in O2 1 ICG001 lungs. (F) Fibronectin gene expression was analyzed by quantitative real-time RT-PCR. Hyperoxia increased fibronectin gene expression in the presence of placebo. Treatment with ICG001 decreased fibronectin gene expression during hyperoxia. *P , 0.05 compared with RA groups; #P , 0.05 compared with O2 1 DMSO (n = 5 group).
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pulmonary vascular remodeling and PH. Treatment with ICG001 significantly decreased in vitro PASMC proliferation under RA and hyperoxia. Decreased cell proliferation in vitro on exposure to hyperoxia as opposed to increased cell proliferation observed in vivo during hyperoxia has been previously reported (56). A “dual hit” mechanism may explain this discrepancy. First, there may be an initial phase of injury in response to oxygen toxicity as observed in short-term in vitro studies, such as ours, where cells are exposed to hyperoxia for 72 hours. During prolonged hyperoxia exposure in vivo, ongoing hyperoxic injury may induce autocrine and paracrine release of cytokines and growth factors that initiate the secondary phase of repair, which is characterized by VSMC and fibroblast proliferation. Therefore, in vivo PASMC proliferation may have occurred in response to hyperoxia after chronic injury and remodeling processes. Although the in vitro culture model lacks the ability to assess the paracrine mechanisms that regulate PASMC proliferation in vivo, the combined in vitro and in vivo data from this study highlight the antiproliferative function of b-catenin signaling inhibition in PASMCs and its role in attenuating pulmonary vascular remodeling. Although b-catenin signaling inhibition resulted in significant reduction in MWT in hyperoxia animals, the effect on PASMC proliferation was only partial. Therefore, antiproliferative effect of ICG001 may only be partially responsible for its anti-remodeling potency and other mechanisms may also be important. Although hyperoxia decreased PASMC proliferation in vitro, the expression of a-SMA, CTGF, and fibronectin was remarkably increased in these cells and was drastically down-regulated by ICG001. These results indicate that hyperoxia induced phenotypical changes in PASMCs via activation of b-catenin signaling. Such phenotypical changes may regulate the pattern of remodeling of the pulmonary vascular bed in response to chronic hyperoxia. Changes in receptor expression or function and in calcium handling may also occur in response to hyperoxia and warrant further investigation. Other possible mechanisms could be involved in b-catenin–mediated hyperoxiainduced pulmonary vascular remodeling and PH. Vascular endothelial cells and
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Figure 6. ICG001 inhibits PASMC proliferation and expression of ECM remodeling molecules in vitro. (A) Proliferation was assessed by Ki67 immunofluorescence at 72 hours. Treatment with ICG001 decreased PASMC proliferation in RA and hyperoxia. **P , 0.01 compared with RA 1 DMSO; ‡‡‡P , 0.001 compared with RA 1 DMSO; †††P , 0.001 compared with RA 1 DMSO (n = 3 per group). (B) Western blot analysis was performed at 24 hours. Relative expression levels of a-SMA, connective tissue growth factor (CTGF), and fibronectin (FN) were normalized by b-actin (housekeeping gene). Lane 1: RA 1 DMSO; lane 2: O2 1 DMSO; lane 3: RA 1 ICG001; Lane 4: O2 1 ICG001. (C) Hyperoxia exposure increased expression of a-SMA, and this was decreased by treatment with ICG001. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO. (D) CTGF expression was significantly increased by hyperoxia in the presence of placebo. Treatment with ICG001 suppressed hyperoxia-induced CTGF expression. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO. (E) Hyperoxia increased FN expression, and this was down-regulated by administering ICG001. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO.
fibroblasts have the ability to proliferate and differentiate into myofibroblasts and migrate into the medial compartments and thus contribute to vascular thickening in chronic hypoxia–induced PH (57). Endothelial cells and fibroblasts may be a direct target of hyperoxia-induced b-catenin–driven nuclear transcription leading to phenotypical and proinflammatory changes. b-Catenin signaling and downstream mediators may also recruit circulating progenitor cells to the adventitial, medial, or intimal compartments, where they assume mesenchymal or even smooth muscle
cell–like characteristics. These changes can directly or indirectly affect underlying PASMCs, thus contributing to chronic abnormalities in vascular tone and structure. Future studies focusing on these mechanisms may provide further insight into the pathogenesis of hyperoxia-induced pulmonary vascular remodeling and PH. There are potential limitations of this study. First, BPD is a multifactorial disease caused by oxygen toxicity, mechanical ventilation, and infection with yet undefined mechanisms. Although high oxygen concentration is needed to induce BPD-like
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pathology in rodents, most infants who develop severe BPD are exposed to lower concentrations of oxygen. It will therefore be important to test the efficacy of ICG001 in larger animal models, such as neonatal baboons or lambs that are exposed to hyperoxia and mechanical ventilation. Second, given the important role of b-catenin signaling in regulating embryonic development and tissue remodeling, inhibition of b-catenin signaling by ICG001 in neonates may induce undesirable side effects. Although we did not observe any side effects on somatic growth (data not shown), it will be crucial to evaluate other developing organs. Third, the dose of ICG001 used in this study was based on published studies in adult animals. However, the pharmacodynamics and pulmonary concentration of ICG001 were not assessed. Future studies are needed to investigate the temporal and spatial effects of ICG001 on inhibition of b-catenin signaling in lungs and in extrapulmonary organs during development under physiological and pathological conditions. In conclusion, our study is the first to describe the successful therapeutic use of a b-catenin signaling inhibitor, ICG001, in a neonatal rat model of hyperoxia-induced BPD and PH. We demonstrate that treatment with ICG001 not only attenuates alveolar simplification provoked by hyperoxia but also ameliorates pulmonary vascular remodeling and PH. The importance of b-catenin signaling in the course of BPD and PH is proven by a significant inhibition of b-catenin signaling by ICG-001. Furthermore, evidence for the involvement of b-catenin signaling in disease development and response to therapy is provided on the cellular and molecular level. Thus, by targeting distinct pathogenic mechanisms, b-catenin signaling inhibitors may have potential as novel therapeutics for severe BPD with PH that will complement currently available standards of care in premature infants. n Author disclosures are available with the text of this article at www.atsjournals.org.
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28. Dasgupta C, Sakurai R, Wang Y, Guo P, Ambalavanan N, Torday J, Rehan V. Hyperoxia-induced neonatal rat lung injury involves activation of TGF-b and Wnt signaling and is protected by rosiglitazone. Am J Physiol Lung Cell Mol Physiol 2009;296: L1031–L1041. 29. Popova AP, Bentley JK, Anyanwu AC, Richardson MN, Linn MJ, Lei J, Wong EJ, Goldsmith AM, Pryhuber GS, Hershenson MB. Glycogen synthase kinase-3b/b-catenin signaling regulates neonatal lung mesenchymal stromal cell myofibroblastic differentiation. Am J Physiol Lung Cell Mol Physiol 2012;303:L439–L448. 30. Chen S, Rong M, Platteau A, Hehre D, Smith H, Ruiz P, Whitsett J, Bancalari E, Wu S. CTGF disrupts alveolarization and induces PH in neonatal mice: implication in the pathogenesis of severe bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 2011;300:L330–L340. 31. Hummler SC, Rong M, Chen S, Hehre D, Alapati D, Wu S. Targeting GSK-3b to prevent hyperoxia-induced lung injury in neonatal rats. Am J Respir Cell Mol Biol 2013;48:578–588. 32. Tanaka A, Jin Y, Lee SJ, Zhang M, Kim HP, Stolz DB, Ryter SW, Choi AM. Hyperoxia-induced LC3B interacts with the Fas apoptotic pathway in epithelial cell death. Am J Respir Cell Mol Biol 2012;46:507–514. 33. Gradl D, Kuhl M, Wedlich D. The Wnt/Wg signal transducer b-catenin controls fibronectin expression. Mol Cell Biol 1999;19:5576–5587. 34. Chiang HY, Korshunov VA, Serour A, Shi F, Sottile J. Fibronectin is an important regulator of flow-induced vascular remodeling. Arterioscler Thromb Vasc Biol 2009;29:1074–1079. 35. Huang BL, Brugger SM, Lyons KM. Stage-specific control of connective tissue growth factor (CTGF/CCN2) expression in chondrocytes by Sox9 and b-catenin. J Biol Chem 2010;285: 27702–27712. 36. Fan WH, Pech M, Karnovsky MJ. Connective tissue growth factor (CTGF) stimulates vascular smooth muscle cell growth and migration in vitro. Eur J Cell Biol 2000;79:915–923. 37. Eguchi M, Nguyen C, Lee SC, Kahn M. ICG-001, a novel small molecule regulator of TCF/beta-catenin transcription. Med Chem 2005;1:467–472. 38. Teo JL, Ma H, Nguyen C, Lam C, Kahn M. Specific inhibition of CBP/ b-catenin interaction rescues defects in neuronal differentiation by a presenilin-1 mutation. Proc Natl Acad Sci USA 2005;102: 12171–12176. 39. Kim H, Won S, Hwang DY, Lee JS, Kim M, Kim R, Kim W, Cha B, Kim T, Costantini F, et al. Downregulation of Wnt/b-catenin signaling causes degeneration of hippocampal neurons in vivo. Neurobiol Aging 2011;32:2316.e1–e15. 40. Yan D, Avtanski D, Saxena NK, Sharma D. Leptin-induced epithelialmesenchymal transition in breast cancer cells requires b-catenin activation via Akt/GSK3-dependent and MTA/Wnt1-dependent pathways. J Biol Chem 2012;287:8598–8612. 41. Alapati D, Rong M, Chen S, Lin C, Li Y, Wu S. Inhibition of LRP5/6-mediated Wnt/b-catenin signaling by Mesd attenuates hyperoxia-induced pulmonary hypertension in neonatal rats. Pediatr Res 2013;73:719–725. 42. Khemani E, McElhinney DB, Rhein L, Andrade O, Lacro RV, Thomas KC, Mullen MP. Pulmonary artery hypertension in formerly premature infants with bronchopulmonary dysplasia: clinical features and outcomes in the surfactant era. Pediatrics 2007;120:1260–1269. 43. Haworth SG. Pulmonary vascular remodeling in neonatal pulmonary artery hypertension, state of art. Chest 1988;93:133S–138S. 44. del Cerro MJ, Rotes AS, Carton A, Deiros L, Bret M, Cordeiro M, Verdu C, Barrios MI, Albajara L, Gutierrez-Larraya F. Pulmonary hypertension in bronchopulmonary dysplasia: clinical findings, cardiovascular anomalies and outcomes. Pediatr Pulmonol 2014;49:49–59. 45. Mourani PM, Abman SH. Pulmonary vascular disease in bronchopulmonary dysplasia: pulmonary hypertension and beyond. Curr Opin Pediatr 2013;25:329–337. 46. Yee M, White RJ, Awad HA, Bates WA, Mcgrath-Morrow SA, O’Reilly MA. Neonatal hyperoxia causes pulmonary vascular disease and shortens life span in aging mice. Am J Pathol 2011;178:2601–2610. 47. Aslam M, Baveja R, Liang OD, Fernandez-Gonzalez A, Lee C, Mitsialis SA, Kourembanas S. None marrow stromal cells attenuate lung injury in a murine model neonatal chronic lung disease. Am J Respir Crit Care Med 2009;180:1122–1130.
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ORIGINAL RESEARCH 48. Ghofrani HA, Seeger W, Grimminger F. Imatinib for the treatment of pulmonary arterial hypertension. N Engl J Med 2005;353: 1412–1413. 49. Frenckner B, Broome M, Lindstrom M, Radell P. Platelet-derived growth factor inhibition: a new treatment of pulmonary hypertension in congenital diaphragmatic hernia? J Pediatr Surg 2008;43: 1928–1931. 50. Uglow EB, Slater S, Sala-Newby GB, Aguilera-Garcia CM, Angelini GD, Newby AC, George SJ. Dismantling of cadherin-mediated cell-cell contacts modulates smooth muscle proliferation. Circ Res 2003;92: 1314–1321. 51. Slater SC, Koutsouki E, Jackson CL, Bush RC, Angelini GD, Newby AC, George SJ. R-cadherin: b-catenin complex and its association with vascular smooth muscle cell proliferation. Arterioscler Thromb Vasc Biol 2004;24:1204–1210. 52. Blidt R, Bosserhoff A-K, Dammers J, Krott N, Demircan L, Hoffmann R, Hanrath P, Weber C, Vogt F. Downregulation of N-cadherin in the neointima stimulates migration of smooth muscle cells by RhoA deactivation. Cardiovasc Res 2004;62:212–222.
53. Jones M, Sabatini PJ, Lee FS, Bendeck MP, Langille BL. Ncadherin upregulation and function in response of smooth muscle cells to arterial injury. Arterioscler Thromb Vasc Biol 2002;22:1972–1977. 54. Quasnichka H, Slater SC, Beeching CA, Boehm M, Sala-Newby GB, George S. Regulation of smooth muscle cell proliferation by b-catenin/TCF signaling involves modulation of cyclin D1 and p21 expression. Circ Res 2006;99:1329–1337. 55. Wang X, Xiao Y, Mou Y, Zhao Y, Blankesteijn M, Hall JL. A role for the b-catenin/TCF signaling cascade in vascular remodeling. Circ Res 2002;90:340–347. 56. Hussain N, Wu F, Christian C, Kresch MJ. Hyperoxia inhibits fetal rat lung fibroblast proliferation and expression of procollagens. Am J Physiol Lung Cell Mol Physiol 1997;273:L726–L732. 57. Arciniegas E, Frid MG, Douglas IS, Stenmark KR. Perspective on endothelial-to-mesenchymal transition: potential contribution to vascular remodeling in chronic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2007;293:L1–L8.
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Abstract Bronchopulmonary dysplasia (BPD) is the most common and serious chronic lung disease of preterm infants. The development of pulmonary hypertension (PH) significantly increases the mortality and morbidity of this disease. b-Catenin signaling plays an important role in tissue development and remodeling. Aberrant b-catenin signaling is associated with clinical and experiment models of BPD. To test the hypothesis that inhibition of b-catenin signaling is beneficial in promoting alveolar and vascular development and preventing PH in experimental BPD, we examined the effects of ICG001, a newly developed pharmacological inhibitor of b-catenin, in preventing hyperoxia-induced BPD in neonatal rats. Newborn rat pups were randomized at postnatal day (P)2 to room air (RA) 1 DMSO (placebo), RA 1 ICG001, 90% FIO2 (O2) 1 DMSO, or O2 1 ICG001. ICG001 (10 mg/kg) or DMSO was given by daily intraperitoneal injection for 14 days during continuous exposure to RA or hyperoxia. Primary human pulmonary arterial smooth muscle cells (PASMCs) were cultured in RA or hyperoxia (95% O2) in the presence of DMSO or ICG001 for 24 to 72 hours. Treatment with ICG001 significantly increased alveolarization and reduced pulmonary vascular remodeling and PH during hyperoxia.
Bronchopulmonary dysplasia (BPD) is the most common and serious chronic lung disease of premature infants (1). Over the past four decades, the incidence of BPD has significantly increased as a result of the improved survival of extremely low-birthweight infants (1–3). There is no effective therapy for BPD due to its multifactorial
Furthermore, administering ICG001 decreased PASMC proliferation and expression of extracellular matrix remodeling molecules in vitro under hyperoxia. Finally, these structural, cellular, and molecular effects of ICG001 were associated with downregulation of multiple b-catenin target genes. These data indicate that b-catenin signaling mediates hyperoxia-induced alveolar impairment and PH in neonatal animals. Targeting b-catenin may provide a novel strategy to alleviate BPD in preterm infants. Keywords: b-catenin; hyperoxia; neonatal lung injury;
bronchopulmonary dysplasia; pulmonary hypertension
Clinical Relevance Inhibition of b-catenin signaling improves alveolarization and reduces pulmonary vascular remodeling and pulmonary hypertension in a neonatal rat model of bronchopulmonary dysplasia. These findings suggest that b-catenin signaling inhibitors may have the potential as novel therapeutics for bronchopulmonary dysplasia.
etiology and poorly understood disease processes. BPD is increasingly being recognized as resulting from interactive mechanisms involving arrested normal lung development and abnormal repair of injury to the immature lung caused by oxygen toxicity, mechanical ventilation, and infection (4, 5). Pulmonary
hypertension (PH) often complicates severe BPD, and this significantly contributes to the mortality and morbidity of preterm infants (6, 7). Studies to investigate the pathogenesis of severe BPD complicated by PH and to identify potential therapeutic targets are urgently needed.
( Received in original form August 1, 2013; accepted in final form January 21, 2014 ) This work was supported by an American Heart Association Grant-in-Aid (S.W.); a Micah Batchelor Award from the Batchelor Foundation (S.W.); by Project NewBorn, University of Miami (S.W.); by the Marta Marx Fund from the Scleroderma Foundation (S.W.); and by Research Award-Ikaria (D.A.). Correspondence and requests for reprints should be addressed to Shu Wu, M.D., Department of Pediatrics/Division of Neonatology, University of Miami Miller School of Medicine, PO Box 016960, Miami, FL 33101. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contenta at www.atsjournals.org Am J Respir Cell Mol Biol Vol 51, Iss 1, pp 104–113, Jul 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0346OC on January 31, 2014 Internet address: www.atsjournals.org
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ORIGINAL RESEARCH b-Catenin is a key regulatory protein with bidirectional capacity to tightly regulate nuclear transcription and to affect cell migration and adhesion by closely interacting with the cytoskeleton and adherens junction cadherins (8–11). A multiprotein “destruction complex” continually phosphorylates b-catenin, flagging it for degradation by the ubiquitin/ proteosome system. Inhibition of this phosphorylation by activation of Wnt and integrin/integrin-linked kinase (ILK) signaling allows b-catenin to accumulate in the cytoplasm and enter the nucleus to form a transcriptional complex localized at gene promoters (12–15). b-Catenin recruits the transcription coactivators, cyclic AMP response-element binding protein (CBP), or its closely related homolog p300 and other components of the basal transcription machinery (16–18). The transcription complex formed by b-catenin and its DNAbinding partners known as lymphocyte enhancer factor/T-cell factor (TCF) is able to promote chromatin remodeling and transcriptional initiation in a cell type–specific and context-specific manner, thus playing a key role in embryonic development, cell proliferation, differentiation, and survival. b-Catenin signaling is one of the important signaling pathways that regulates normal lung development and homeostasis (19, 20). Aberrant b-catenin signaling is associated with abnormal epithelial proliferation, fibroproliferative repair, and lung matrix remodeling in response to acute and chronic lung injuries (21–24). In fact, inhibition of b-catenin transcription activity by ICG001, a newly developed small molecule, ameliorated and reversed pulmonary fibrosis in a mouse model of bleomycin-induced lung injury (25, 26). We and others have previously demonstrated that b-catenin signaling is increased in the lungs of hyperoxia-exposed neonatal rats, a widely used experimental model of BPD (27, 28). A recent study demonstrated that increased b-catenin is present in the mesenchymal stromal cells isolated from infants developing BPD and in a-smooth muscle actin (a-SMA)-positive myofibroblasts in the lungs of infants dying of BPD (29). Together, these data suggest that aberrant b-catenin signaling may play an important role in the pathogenesis of BPD. In the current study, we tested the hypothesis that inhibition of b-catenin
signaling is beneficial in promoting alveolar and vascular development and preventing PH in a hyperoxia-induced severe BPD model in neonatal rats. We demonstrate that treatment with ICG001 improves alveolar and vascular development, decreases pulmonary vascular remodeling, and reduces PH during hyperoxia. In cultured human pulmonary artery smooth muscle cells (PASMCs), treatment with ICG001 decreases cell proliferation and inhibits expression of a-SMA, fibronectin, and connective tissue growth factor (CTGF) under hyperoxia exposure. Thus, targeting b-catenin signaling may provide a novel strategy for preventing and treating BPD in preterm infants.
Materials and Methods Detailed descriptions of the materials and methods are provided in the online supplement. Animal Models
The animal protocol was approved by the University of Miami Institutional Animal Care and Use Committee. Newborn Sprague Dawley rat pups were randomized at postnatal day (P)2 to room air (RA) 1 DMSO (placebo), RA 1 ICG001 (a specific pharmacological inhibitor of b-catenin), 90% FIO2 (O2) 1 DMSO, and O2 1 ICG001. ICG001 (10 mg/kg) or DMSO (99%, equal volume) was given by daily intraperitoneal injection for 14 days during continuous RA or hyperoxia exposure. Hemodynamic Measurements
On P15, right ventricular systolic pressure (RVSP) and right ventricle (RV) to left ventricle (LV) plus septum (LV 1 S) weight ratio (RV/LV 1 S) were determined as indices for PH (30). Briefly, rats were sedated, tracheotomized, and ventilated with a Mini-Vent (Harvard Apparatus, Holliston, MA). After thoracotomy, a 25-gauge needle fitted to a pressure transducer was inserted into the RV. RVSP was measured and continuously recorded on a Gould polygraph (model TA-400; Gould Instruments, Cleveland, OH). Immediately after RVSP measurements, hearts were dissected for RV free wall separation from LV1S for RV/LV 1 S weight ratio measurements.
Alapati, Rong, Chen, et al.: b-Catenin Signaling in Experimental BPD
Lung Histology and Morphometry
Lungs were infused with 4% paraformaldehyde via a tracheal catheter at 20 cm H2O of pressure for 5 minutes, fixed overnight, and paraffin embedded. Hematoxylin and eosin–stained tissue sections were used to measure mean linear intercept (MLI), septal density, and radial alveolar count as previously described (27, 30, 31). Immunofluorescence and Double Immunofluorescence Staining
Immunofluorescence and double immunofluorescence staining were performed as previously described (27, 30). Pulmonary Vascular Morphometry
Pulmonary vascular density was determined by the average number of vonWillebrand factor–stained vessels (, 50 mm in diameter) from 10 random images on each lung section (27, 30). Assessment of Pulmonary Vascular Remodeling
Immunofluorescence staining for a-SMA was performed, and 20 peripheral pulmonary vessels (, 50 mm in diameter) were assessed for medial wall thickness (MWT) (27, 30). Proliferation of vascular smooth muscle cells (VSMCs) was assessed by double immunofluorescence staining for a-SMA and Ki67, a proliferation marker on lung sections. Expression of fibronectin was determined by immunofluorescence staining and real-time quantitative RT-PCR (qRT-PCR). Western Blot Analysis
Total protein extraction and Western blot analysis were performed as previously described (27, 30). Real-Time qRT-PCR
Total RNA isolation and real-time qRT-PCR was performed as previously described (30). PASMC Culture and Treatment
Human PASMCs were cultured according to the manufacturer’s instruction (Lonza, Portsmouth, NH) and used between passages 2 through 6. Cells were cultured in 10% FCS containing media to 60 to 70% confluence and then cultured in 1% FCS containing media for 24 hours. The cells were treated with ICG001 (10 mM) or DMSO and exposed to RA or 95% O2 for 24 to 72 hours in a sealed plastic chamber as previously described (32). 105
ORIGINAL RESEARCH Immunofluorescence staining for Ki67 was performed in cells grown on chamber slides. Cell proliferation index was determined by the ratio of Ki67-positive nuclei to total nuclei. Proteins were isolated from cells grown in 6-well plates for subsequent Western blot analysis. Statistical Analysis
Data were expressed as means 6 SD. Comparison among the groups was performed by one-way ANOVA followed by Student-Newman Keuls test. A P value , 0.05 was considered significant.
Results ICG001 Inhibits Hyperoxia Activation of b-Catenin Signaling In Vivo
We first examined b-catenin signaling by assessing b-catenin localization. Hyperoxia exposure induced diffuse b-catenin nuclear translocation in lungs treated with placebo or ICG001 (Figure 1A). Double immunofluorescence colocalized some of the b-catenin–positive nuclei in surfactant protein C (SP-C)–expressing cells, suggesting that b-catenin undergoes nuclear translocation in alveolar type II epithelial cells (Figure 1A). There was no significant difference in the index of b-catenin nuclear translocation between the hyperoxia plus placebo and hyperoxia plus ICG001 groups (Figures 1A and 1B). We then assessed expression of Wntinduced signaling protein-1 (WISP-1), a well-characterized b-catenin target gene. Hyperoxia exposure significantly increased WISP-1 expression in the placebo-treated lungs, and this was decreased by administration of ICG001 during hyperoxia (Figure 1C). Inhibition of b-Catenin Signaling Improves Alveolar development
Because hyperoxia exposure is known to impair alveolarization, we evaluated the effect of ICG001 on alveolar development by histology and morphometry. On histological examination, the lungs from the hyperoxia plus placebo group displayed large and simplified alveoli with fewer secondary septa (Figure 2A). Treatment with ICG001 improved alveolarization in hyperoxiaexposed lungs, which now appeared similar to RA-exposed lungs (Figure 2A). Morphometric analysis demonstrated 106
Figure 1. ICG001 inhibits b-catenin signaling. (A) Immunofluorescence staining for b-catenin (red signal), pro-surfactant C (pro-SP-C) (green signal), and DAPI nuclear staining (blue signal) were performed on lung sections from neonatal rats exposed to room air (RA) plus DMSO (RA 1 DMSO), RA 1 ICG001, hyperoxia plus DMSO (O2 1 DMSO), and O2 1 ICG001. Pink signals indicate colocalization of b-catenin with nuclei. Arrow indicates SP-C–expressing cells. In hyperoxia-exposed lungs, some of the b-catenin–positive nuclei were colocalized in pro-SPC–expressing cells. The b-catenin nuclear translocation (NT) index (% of b-catenin positive nuclei/total nuclei) was determined (B). Hyperoxia exposure in the presence of DMSO increased the b-catenin NT index compared with RA groups, and this was not affected by administration of ICG001. ***P , 0.001 compared with RA groups; †††P , 0.001 compared with RA groups (n = 5 per group). (C) Western blot analysis demonstrated that hyperoxia exposure increased Wntinduced signaling protein-1 (WISP1) expression in the placebo lungs, and this was significantly decreased by ICG001 treatment. Lane 1: RA 1 DMSO; lane 2: O2 1 DMSO; lane 3: RA 1 ICG001; lane 4: O2 1 ICG001. *P , 0.05 compared with RA groups; #P , 0.05 compared with O2 1 DMSO (n = 5 per group).
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ORIGINAL RESEARCH we next evaluated the effect of ICG001 on RVSP and RV/LV1S weight ratio as indices of PH and right ventricular hypertrophy (RVH), respectively. Compared with RA groups, the rats in the hyperoxia plus placebo group had significantly increased RVSP (Figure 4A) and RVH (Figure 4B). Administration of ICG001 during hyperoxia significantly decreased RVSP (17.80 6 2.56 versus 25.09 6 3.15, P , 0.001; O2 1 ICG001 versus O2 1 DMSO) and RV/LV1S weight ratio (0.34 6 0.05 versus 0.58 6 0.10, P , 0.001; O2 1 ICG001 versus O2 1 DMSO) (Figures 4A and 4B). These findings indicate that inhibition of b-catenin signaling protects against hyperoxia-induced PH in neonatal rats. Inhibition of b-Catenin Signaling Decreases Pulmonary Vascular Remodeling
Figure 2. Inhibition of b-catenin improves alveolarization. (A) Lung histology. Hyperoxia exposure increased mean linear intercept (MLI) (B), decreased septal density (C), and decreased radial alveolar count (RAC) (D) in the presence of DMSO as compared with RA. Administration of ICG001 decreased MLI, increased septal density, and increased RAC during hyperoxia. **P , 0.01 and ***P , 0.001 compared with RA; #P , 0.05 and ###P , 0.001 compared with O2 1 DMSO; † P , 0.05 compared with RA groups (n = 5 per group).
a significant increase in MLI in the hyperoxia lungs treated with the placebo as compared with the RA plus placebo group (51.23 6 3.06 versus 39.35 6 0.68, P , 0.001; O2 1 DMSO versus RA 1 DMSO) (Figure 2B), signifying larger and fewer alveoli. In contrast, treatment with ICG001 during hyperoxia significantly decreased MLI as compared with the corresponding hyperoxia group that received placebo (44.26 6 0.75 versus 51.23 6 3.06, P , 0.05; O2 1 ICG001 versus O2 1 DMSO) (Figure 2B). Correlating with decreased MLI, administration of ICG001 significantly increased secondary septal density as compared with placebo during hyperoxia (Figure 2C). Radial alveolar count was also significantly increased by ICG001 in hyperoxia-exposed lungs (Figure 2D). Thus, inhibition of b-catenin
signaling improves alveolar development during hyperoxia. Inhibition of b-Catenin Signaling Improves Vascular Development
Because hyperoxia exposure can inhibit pulmonary vascular development, we assessed vascular density in these lungs. Compared with RA-exposed lungs, hyperoxia exposure significantly decreased vascular density in the presence of the placebo (Figures 3A and 3B). Treatment with ICG001 modestly increased vascular density during hyperoxia as compared with the hyperoxia plus placebo group (Figures 3A and 3B). Inhibition of b-Catenin Signaling by ICG001 Reduces PH
Our previous studies have shown that hyperoxia exposure induces PH. Therefore,
Alapati, Rong, Chen, et al.: b-Catenin Signaling in Experimental BPD
Given that ICG001 attenuated hyperoxiainduced PH, we sought to elucidate whether inhibition of b-catenin signaling decreases pulmonary vascular remodeling. The degree of muscularization of peripheral pulmonary arterioles (, 50 mm in diameter) was determined by measuring the MWT on a-SMA–stained lung sections. The lungs in the hyperoxia plus placebo group had a significant increase in MWT compared with the RA plus placebo group (0.46 6 0.05 versus 0.21 6 0.03, P , 0.001; O2 1 DMSO versus RA 1 DMSO) (Figures 5A and 5B). However, treatment with ICG001 during hyperoxia decreased MWT to near RA levels (0.26 6 0.03 versus 0.46 6 0.05, P , 0.001; O2 1 ICG001 versus O2 1 DMSO). These data indicate that inhibition of b-catenin signaling prevents hyperoxia-induced excessive muscularization of peripheral pulmonary vasculature. Because abnormally increased proliferation of PASMCs contributes partly to increased muscularization of peripheral pulmonary arterioles in response to chronic lung injury, we assessed proliferation of PASMCs in Ki67 and a-SMA doublestained lung tissue sections. There were increased Ki67-positive PASMCs in the medial wall of peripheral pulmonary arterioles in the hyperoxia- and placeboexposed lungs as compared with RA groups (Figures 5C and 5D). In contrast, treatment with ICG001 significantly decreased the proliferation index of PASMCs as compared with the hyperoxia plus placebo group (18.20 6 5.41 versus 30.40 6 8.93, 107
ORIGINAL RESEARCH among the study groups (data not shown). Thus, ICG001 exerts direct antiproliferative effects on PASMCs and may contribute to the decreased proliferation of PASMCs observed in vivo. Expression of a-SMA in cultured PASMCs was markedly increased by hyperoxia exposure (0.72 6 0.01 versus 0.37 6 0.05, P , 0.001; O2 1 DMSO versus RA 1 DMSO) (Figure 6C). However, treatment of hyperoxia-exposed PASMCs with ICG001 decreased a-SMA expression to RA levels (0.28 6 0.01 versus 0.72 6 0.01, P , 0.001; O2 1 ICG001 versus O2 1 DMSO) (Figure 6C). CTGF and fibronectin are b-catenin target genes that play an important role in ECM deposition and in vascular remodeling (33–36). We thus evaluated whether inhibition of b-catenin signaling would down-regulate CTGF and fibronectin expression in PASMCs. Treatment with ICG001 significantly decreased expression of fibronectin and CTGF under hyperoxia (Figures 6D and 6E). Taken together, these findings highlight that b-catenin signaling plays a critical role in PASMC proliferation, differentiation, and remodeling in vitro.
Discussion Figure 3. Inhibition of b-catenin improves vascular development. (A) Immunofluorescence staining for vonWillebrand factor (vWF) (red signal) and DAPI nuclear staining (blue signal) was performed. (B) Vascular density (VD) was determined by counting vWF-positive vessels (, 50 mm) on 10 random high-power-field (HPF) images from each lung section. Hyperoxia exposure in the presence of the placebo significantly decreased VD as compared with RA groups. Administration of ICG001 significantly increased VD in hyperoxia-exposed lungs; however, it was still less than the RA groups. ***P , 0.001 compared with RA groups; †††P , 0.001 compared with RA groups; #P , 0.05 compared with O2 1 DMSO (n = 5–6 per group).
P , 0.01; O2 1 ICG001 versus O2 1 DMSO). Thus, inhibition of b-catenin signaling antagonizes hyperoxia-induced PASMC proliferation in neonatal lungs. Fibronectin is a b-catenin target gene that plays an important role in vascular remodeling (33, 34). We therefore assessed fibronectin protein and gene expression. In hyperoxia- and placebo-exposed lungs, there was increased fibronectin protein expression in the peripheral pulmonary vessels as compared with the lungs exposed to RA. Administration of ICG001 decreased vascular fibronectin expression during hyperoxia (Figure 5E). RT-qPCR confirmed that fibronectin gene expression was increased by hyperoxia and decreased by ICG001 treatment (Figure 5F). 108
Effects of ICG001 on PASMC Proliferation and Expression of ECM Remodeling Molecules In Vitro
Having observed the protective effects of ICG001 on PH and pulmonary vascular remodeling, we sought to further determine the effects of ICG001 on PASMC proliferation and expression of ECM remodeling molecules in vitro. Hyperoxia exposure in the presence of the placebo decreased PASMC proliferation (Figure 6A). Treatment with ICG001 decreased PASMC proliferation under RA and hyperoxia (Figure 6A). We also assessed cell apoptosis by examining expression of cleaved caspase-3 and found that there was no significant difference
In the present study, we demonstrate that hyperoxia exposure activates b-catenin signaling in the lungs of neonatal rats. Treatment with ICG001 inhibits b-catenin signaling and improves alveolarization and pulmonary vascular development in these animals. Inhibition of b-catenin signaling also drastically reduces RVSP and RVH, indicating decreased PH. The improvement in PH is associated with decreased proliferation of PASMCs in the medial wall of peripheral pulmonary vessels and a concomitant decrease in the MWT. In cultured PASMCs, inhibition of b-catenin signaling decreases cell proliferation and differentiation and down-regulates expression of the genes responsible for ECM remodeling. Collectively, these data suggest that inhibition of b-catenin signaling may interfere with the progression of hyperoxiainduced PH and pulmonary vascular remodeling through a direct inhibitory effect on PASMCs. This study therefore highlights the importance of b-catenin signaling in the pathogenesis of PH in a hyperoxiainduced rodent model of severe BPD and provides a potential therapeutic target for alleviating PH in infants with severe BPD.
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Figure 4. Inhibition of b-catenin decreases pulmonary hypertension. (A) Right ventricular systolic pressure (RVSP) was significantly increased in the O2 1 DMSO lungs as compared with RA groups. ICG001 administration significantly decreased RVSP during hyperoxia. ***P , 0.001 compared with the RA groups; †††P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO (n = 6–11 per group). (B) Right ventricular hypertrophy (RVH) was determined by the weight ratio of right ventricle to left ventricle 1 septum. Hyperoxia-exposed animals in the presence of the placebo presented with increased RVH, and this was significantly decreased by ICG001 treatment. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO (n = 6–11 per group).
b-Catenin normally controls cell–cell adhesion through its binding with cadherin in adherens junctions and mediates the link between cadherin and the actin cytoskeleton (8–11). In addition, b-catenin acts as a transcriptional activator and regulates transcription of target genes responsible for cell proliferation, apoptosis, and differentiation (12, 13). b-Catenin nuclear translocation can be activated by Wnt signaling and by integrin/ILK signaling (12–15). Within the nucleus, b-catenin forms a transcription complex with its DNA-binding partners lymphocyte enhancer factor/TCF. In this complex, b-catenin serves as an obligate coactivator through its ability to recruit CBP to promote chromatin remodeling and transcription initiation/elongation (16–18). ICG001 interacts with CBP and specifically blocks the b-catenin/CBP interface and down-regulates b-catenin/TCF signaling (25, 26, 37–39). Previous studies have demonstrated the efficacy of ICG001 in treating bleomycin-induced lung fibrosis in adult mice (26) and in blocking epithelial–mesenchymal transition in vitro (40). Hyperoxia-induced lung injury in neonatal rats has been widely used as an experimental model of BPD for mechanistic studies and therapeutic exploration. We and others have previously shown that chronic hyperoxia exposure increases b-catenin nuclear translocation and further signaling in the lungs of neonatal rats (27, 28, 41). In the present study, we confirmed that exposure to 90% O2 for 14 days increased b-catenin nuclear translocation and target gene expression and showed for the first time that inhibition of b-catenin–driven
nuclear transcription by ICG001 prevents hyperoxia-induced alveolar simplification. This is consistent with our previous findings that prevention of hyperoxiainduced b-catenin nuclear translocation by a neutralizing monoclonal antibody for CTGF resulted in a significant attenuation of hyperoxia-induced alveolar injury in neonatal rats (27). CTGF has the ability to activate b-catenin signaling through binding to Wnt coreceptor LRP5/6 and cell surface integrins. Although we have also previously showed that inhibition of Wnt receptor LRP5/6 by Mesd does not improve alveolarization during hyperoxia (41), Dasqupta and colleagues have previously shown that treatment with rosiglitazone, an agonist of peroxisome proliferator-activated receptor-g, protects against hyperoxiainduced alveolar damage by decreasing TGF-b and Wnt/b-catenin signaling (28). It is plausible to suggest that disruption of alveolarization by hyperoxia could be independent of LRP5/6 and that multiple other signaling pathways, such as integrin/ ILK and peroxisome proliferator-activated receptor-g, may play a more prominent role in b-catenin activation and alveolar development under hyperoxia. Nevertheless, this study signifies the critical role of b-catenin signaling in the pathogenesis of neonatal hyperoxia– induced alveolar impairment. The novel finding of the present study is that inhibition of b-catenin signaling significantly attenuates hyperoxia-induced PH. The reduced PH we observed was associated with a dramatic decrease in pulmonary vascular remodeling and a modest improvement in vascular density. This is important because PH contributes
Alapati, Rong, Chen, et al.: b-Catenin Signaling in Experimental BPD
significantly to the mortality and morbidity of preterm infants with severe BPD (6, 7). Despite the widespread availability and use of pulmonary vasodilators such as nitric oxide, the mortality rate of infants with BPD complicated by PH remains as high as 50% (42). This high mortality rate is associated with the pulmonary vascular changes observed in these patients. These include increased vascular tone and reactivity, hypertensive vascular remodeling, and decreased vascular growth (43–45). BPD is a multifactorial disease, and oxygen toxicity is one of the important causes. Although intermittent hypoxia has been shown to directly contribute to PH, extensive animal studies have demonstrated that hyperoxia exposure independently contributes to the vascular changes observed in BPD (46, 47). Therapeutic agents that target distinct pathogenic mechanisms of pulmonary vascular remodeling and growth in severe BPD may provide additional relief on PH and decrease mortality. As such, recent clinical trials have reported better results in adult PH using therapeutic agents that reversed lung vascular remodeling rather than prolonged vasodilatation (48, 49). Thus, targeting b-catenin signaling to alleviate pulmonary vascular remodeling may be a promising new therapy for PH in severe BPD. The dynamic process of vascular remodeling involves numerous molecular signaling cascades governing VSMC migration, differentiation, proliferation, and fate. Studies that focused on elucidating the underlying pathogenetic mechanisms of atherosclerosis and graft rejection implicated the cadherin family of cell adhesion molecules as having an important role in VSMC proliferation (50–53). Quasnicka and colleagues demonstrated that growth factors such as PDGF and bFGF that modulate human aortic VSMC proliferation do so via the activation of b-catenin/TCF signaling (54). Wang and colleagues also demonstrated that after balloon injury there were increased b-catenin expression and vascular remodeling in rat carotid artery VSMCs (55). In the same study, the authors described a dual role of b-catenin/TCF signaling in inhibiting apoptosis and promoting proliferation in VSMCs. We therefore used PASMC cultures to elucidate the underlying mechanisms by which inhibition of b-catenin signaling reduces 109
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Figure 5. Effects of ICG001 on pulmonary vascular remodeling. (A) Immunofluorescence staining for a-SMA (green signal) and DAPI nuclear staining (blue signal). (B) Medial wall thickness (MWT) was assessed from 20 peripheral vessels (, 50 mm in diameter) on each lung section. Hyperoxia increased MWT in the presence of placebo as compared with RA groups. ICG001 administration significantly decreased MWT in hyperoxiaexposed lungs. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO (n = 5 per group). (C) Double immunofluorescence staining for a-SMA (green signal), Ki67 (red signal), and DAPI nuclear staining (blue signal). (D) The PASMC proliferative index was determined as the percentage of Ki67positivea-SMApositive/a-SMApositive cells in vessels that are , 50 mm in diameter. Hyperoxia increased the PASMC proliferative index when exposed to the placebo. Treatment with ICG001 modestly decreased the PASMC proliferative index during hyperoxia. ***P , 0.001 compared with the RA groups; ††P , 0.01 compared with RA groups; ##P , 0.01 compared with O2 1 DMSO (n = 5 per group). (E) Immunofluorescence staining for fibronectin (green signal) and DAPI nuclear staining (blue signal). Fibronectin staining was increased in O2 1 DMSO lungs, and this was decreased in O2 1 ICG001 lungs. (F) Fibronectin gene expression was analyzed by quantitative real-time RT-PCR. Hyperoxia increased fibronectin gene expression in the presence of placebo. Treatment with ICG001 decreased fibronectin gene expression during hyperoxia. *P , 0.05 compared with RA groups; #P , 0.05 compared with O2 1 DMSO (n = 5 group).
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pulmonary vascular remodeling and PH. Treatment with ICG001 significantly decreased in vitro PASMC proliferation under RA and hyperoxia. Decreased cell proliferation in vitro on exposure to hyperoxia as opposed to increased cell proliferation observed in vivo during hyperoxia has been previously reported (56). A “dual hit” mechanism may explain this discrepancy. First, there may be an initial phase of injury in response to oxygen toxicity as observed in short-term in vitro studies, such as ours, where cells are exposed to hyperoxia for 72 hours. During prolonged hyperoxia exposure in vivo, ongoing hyperoxic injury may induce autocrine and paracrine release of cytokines and growth factors that initiate the secondary phase of repair, which is characterized by VSMC and fibroblast proliferation. Therefore, in vivo PASMC proliferation may have occurred in response to hyperoxia after chronic injury and remodeling processes. Although the in vitro culture model lacks the ability to assess the paracrine mechanisms that regulate PASMC proliferation in vivo, the combined in vitro and in vivo data from this study highlight the antiproliferative function of b-catenin signaling inhibition in PASMCs and its role in attenuating pulmonary vascular remodeling. Although b-catenin signaling inhibition resulted in significant reduction in MWT in hyperoxia animals, the effect on PASMC proliferation was only partial. Therefore, antiproliferative effect of ICG001 may only be partially responsible for its anti-remodeling potency and other mechanisms may also be important. Although hyperoxia decreased PASMC proliferation in vitro, the expression of a-SMA, CTGF, and fibronectin was remarkably increased in these cells and was drastically down-regulated by ICG001. These results indicate that hyperoxia induced phenotypical changes in PASMCs via activation of b-catenin signaling. Such phenotypical changes may regulate the pattern of remodeling of the pulmonary vascular bed in response to chronic hyperoxia. Changes in receptor expression or function and in calcium handling may also occur in response to hyperoxia and warrant further investigation. Other possible mechanisms could be involved in b-catenin–mediated hyperoxiainduced pulmonary vascular remodeling and PH. Vascular endothelial cells and
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Figure 6. ICG001 inhibits PASMC proliferation and expression of ECM remodeling molecules in vitro. (A) Proliferation was assessed by Ki67 immunofluorescence at 72 hours. Treatment with ICG001 decreased PASMC proliferation in RA and hyperoxia. **P , 0.01 compared with RA 1 DMSO; ‡‡‡P , 0.001 compared with RA 1 DMSO; †††P , 0.001 compared with RA 1 DMSO (n = 3 per group). (B) Western blot analysis was performed at 24 hours. Relative expression levels of a-SMA, connective tissue growth factor (CTGF), and fibronectin (FN) were normalized by b-actin (housekeeping gene). Lane 1: RA 1 DMSO; lane 2: O2 1 DMSO; lane 3: RA 1 ICG001; Lane 4: O2 1 ICG001. (C) Hyperoxia exposure increased expression of a-SMA, and this was decreased by treatment with ICG001. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO. (D) CTGF expression was significantly increased by hyperoxia in the presence of placebo. Treatment with ICG001 suppressed hyperoxia-induced CTGF expression. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO. (E) Hyperoxia increased FN expression, and this was down-regulated by administering ICG001. ***P , 0.001 compared with RA groups; ###P , 0.001 compared with O2 1 DMSO.
fibroblasts have the ability to proliferate and differentiate into myofibroblasts and migrate into the medial compartments and thus contribute to vascular thickening in chronic hypoxia–induced PH (57). Endothelial cells and fibroblasts may be a direct target of hyperoxia-induced b-catenin–driven nuclear transcription leading to phenotypical and proinflammatory changes. b-Catenin signaling and downstream mediators may also recruit circulating progenitor cells to the adventitial, medial, or intimal compartments, where they assume mesenchymal or even smooth muscle
cell–like characteristics. These changes can directly or indirectly affect underlying PASMCs, thus contributing to chronic abnormalities in vascular tone and structure. Future studies focusing on these mechanisms may provide further insight into the pathogenesis of hyperoxia-induced pulmonary vascular remodeling and PH. There are potential limitations of this study. First, BPD is a multifactorial disease caused by oxygen toxicity, mechanical ventilation, and infection with yet undefined mechanisms. Although high oxygen concentration is needed to induce BPD-like
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