PM2.5 Induced Airway Remodeling via Wnt5a/β- catenin Pathway in Chronic Obstructive Pulmonary Diseases

Weifeng Zou Guangzhou Chest Hospital https://orcid.org/0000-0002-6514-0704 Xiaoqian Wang Mianyang Central Hospital Ruiting Sun Guangzhou Medical University Jinxing Hu Guangzhou Chest Hospital Dong Ye Guangzhou Medical University Sha Liu University of South China Meihua Guo Guangzhou Chest Hospital Pixin Ran (  pxran@gzhmu.edu.cn ) The First A liated Hospital of Guangzhou Medical University https://orcid.org/0000-0001-6651-634X

Conclusions: Our ndings suggest that PM2.5 exposure induce HBSMC proliferation, contributing to airway remodeling via Wnt5a/β-Catenin signaling pathway in vivo and in vitro, which could be a target of treatment of COPD.

Background
Chronic obstructive pulmonary disease (COPD) is a common chronic disease that can be prevented and treated. It is accompanied by persistent respiratory symptoms and air ow limitation, generally with airway and alveolar abnormalities related to the signi cantly exposure to harmful particles or gases [1][2][3].
Airway remodeling is a distinctive pathological feature of COPD, and airway wall thickness correlates with the severity of COPD. Smooth muscle cell proliferation plays an important role in airway remodeling, and the smooth muscle layer of the small airways is thickened and correlated negatively with the severity of the disease in patients with COPD [4][5][6]. Although there are advances in the understanding of the in ammatory features of COPD, the mechanisms underlying the remodeling process are still poorly understood.
PM2.5 is an ambient air pollutant with a diameter of ≤2.5 μm, characterized by small particle size, large surface area, and the ability to absorb toxins [7]. Recently, PM2.5 exposure is associated not only with an enhanced risk of developing COPD, but also with the exacerbated deterioration of lung function and symptoms experienced by COPD patients [8,9]. We previously showed that PM2.5 induced the expression of Wnt5a in human bronchial epithelial cells [10]. Wnt5a belongs to the Wnt gluco-protein family which activates a variety of downstream signaling pathways to regulate cell migration, polarity, proliferation and survival [11]. Previous studies have shown that increased expression of Wnt5a activated nonclassical Wnt signaling contributes to the pathogenesis of COPD [12]. Speci cally, Wnt5a increased TGF-β1-induced upregulation of α-SMA expression to enhance smooth muscle cells contractility [13]. TGF-β1 is involved in β-catenin-independent non-classical Wnt5a signaling, which induces ECM expression in airway smooth muscle cells and promotes tissue brosis [14]. However, Wnt5a regulates the proliferation of smooth muscle cells and its role in regulation of airway remodeling in response to PM2.5 remains unknown.
Platelet-derived growth factor receptor (PDGFRβ) acts as a cell surface tyrosine kinase receptor and is involved in cell proliferation, survival and migration [15]. Emerging evidence suggests that Wnt signaling can contribute to smooth muscle cell precursor proliferation and adult lung disease development by directly regulating TenascinC transcription participating in the regulation of PDGFRβ, which de ned the Wnt/Tnc/PDGFR signaling axis [16]. However, it remains unclear whether this axis promotes airway remodeling and accelerates disease progression in COPD.
On this basis, we hypothesized that the Wnt5a/β-Catenin signaling pathway is participated in PM2.5induced airway smooth muscle cell proliferation through upregulation of PDGFRβ expression, and led to airway remodeling. Hence, using HBSMC and an experimental model of COPD, this study was designed to elucidate the effects of Wnt/Tnc / PDGFRβ axis in the airway remodeling of COPD caused by PM2.5.

Methods
Animal Experiments C57BL/6 mice were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. Animal experiments were performed following the Declaration of Helsinki Convention on the Use and Care of Animals and were approved by the Animal Research Ethics Committee of Guangzhou Medical University.
Collection and Extraction of PM2.5 Tra c ambient PM2.5 was collected by aerodynamic impactors equipped with a glass ber lter, quartz lter, or te on membrane, DMSO was used to extract soluble parts of PM2.5. Su cient speci c samples for biological experiments were collected by high volume impactors equipped with glass ber lter from arterial tra c road. Gravimetric analysis was used to analyze speci c physicochemical characterizations of PM2.5. The procedure of PM2.5 samples and analysis methods were obtained as our previously described [10].

Histology and Immunohistochemistry
Mice lung tissues were collected and analyzed for small airways by histochemistry and immunohistochemistry according to our previous descriptions [17,18]. The histology was performed on 4.0 μm thick para n sections of lung tissues. Sections were incubated with Anti-α-SMA antibody.

Analysis method of airway remodeling
The thickness of the small airway wall was the total area of the small airway wall (WAt) after normalization of the perimeter of basement membrane(Pbm), i.e., WAt/Pbm (μm 2 /μm), which was used as an index of small airway remodeling. The requirement of airway was selected as follows: 1. basement membrane perimeter < 2000 μm; 2. minimum internal diameter of airway/maximum internal diameter of airway > 0.5; Pbm, area of basement membrane(Abm) and airway wall outer membrane area (Ao) were measured using Image pro-plus 6.0 (IPP6.0) software; The percentage of smooth muscle layer area (smooth muscle speci c marker protein α-SMA positive area) to airway wall area, i.e. Area/(Ao-Abm)% (WA%), was used as an indicator of smooth muscle layer in airway remodeling.

Analysis method of emphysema
The mean linear intercept (MLI) was used to evaluate the size of the alveolar cavity, which was used as an index of emphysema. At the same eld of view, " " intersecting lines were drawn, and the length of each line (L) and the number of alveoli passing through each line (NS) were calculated, and MLI was expressed as MLI=L/NS.

Cell culture and treatment
The human bronchial smooth muscle cell line (HBSMC; American Type Culture Collection, Manassas, VA, USA) was cultured in SMCM (sciencell, USA). Cells were cultured at 37°C with 5% CO 2 with humidi cation and then exposed (or not) to PM2.5 (3 μg/ml). HBSMC were pretreated with the Wnt5a antagonist BOX5 (Merck Millipore, Burlington, MA, USA) for 1 h prior to the addition of 3 μg/ml PM2.5.

Isolation of mRNA and real-time PCR analysis
Total mRNA was extracted using the Universal RNA Extraction Kit (B0004D, EZB, Shanghai, China) according to the manufacturer's instructions. mRNA was quanti ed using a NanoDrop spectrophotometer (NanoDrop Tech, Rockland, DE, USA). Gene-speci c primers (listed in Table 1) were provided by Sangon Biotech (Shanghai, China). Quantitative real-time PCR was performed using a SYBR Premix Ex Taq II (Novozymes) and a Bio-rad instrument II (Roche Diagnostics, Basel, Switzerland). GAPDH served as the housekeeping control. Table 1 Primer Primer sequence Human Wnt5a F:5'-ATTCTTGGTGGTCGCTAGGTA-3'

Statistical analysis
All statistical analyses were performed using GraphPad Prism 7.04 for windows (GraphPad Software, San Diego, USA) and are presented as median and IQR. Data was analyzed with a one-way ANOVA with Bonferroni post-hoc test or Kruskal-Wallis with Dunn's test for multiple comparison depending on respectively parametric or non-parametric datasets. p<0.05 was considered statistically signi cant.
Result PM2.5 stimulation induced airway remodeling and emphysema in mice.
We used H & E staining to observe the changes of lung histology and immunohistochemistry to detect the expression of α-SMA. Compared with the control group, the lung tissue destruction in different concentration of PM2.5-exposed mice was obvious, showing obvious emphysematous features such as enlarged and fused alveolar cavities and thinning, breaking and widening of alveolar septa (Fig 1A and  1B), indicating the occurrence of emphysema-like lesions. Lung function tests showed that different concentration of PM2.5 exposure downregulated the level of Peak inspiratory ow (PIF) and Peak inspiratory ow (PEF) in mice (Fig 1C and 1D). Based on the structural changes of alveoli, lung function and survival rate of mice, the mice in the medium concentration of PM2.5 exposure group were selected for follow-up experiments with reference to relevant literature. Compared with the control group, the airway wall thickness of mice exposed to PM2.5 for 1 month was signi cantly thickened (Fig 1E and 1F). Furthermore, the PM2.5 group showed increased α-SMA expression, thickening of the smooth muscle layer, and the percentage of the smooth muscle layer in the airway wall was signi cantly increased ( Fig  1G-1I), suggesting that PM2.5 induced small airway remodeling in vivo.

PM2.5 induced HBSMC proliferation
To investigate the effect of PM2.5 on airway smooth muscle, we detected the expression of α-SMA and Proliferating cell nuclear antigen (PCNA) in airway smooth muscle cells after PM2.5 stimulation. PCNA is an intranuclear protein and involved in DNA replication that is mainly produced in proliferating and transformed cells [19]. After PM2.5 stimulation of airway smooth muscle cells, PCNA and α-SMA expression were upregulated (Fig 2A-2C), indicating that PM2.5 promoted airway smooth muscle proliferation.
PM2.5 increased Wnt5a/β-Catenin pathway activation in lung tissue of mice To investigate the effect of PM2.5 on the expression of Wnt5a/β-Catenin signaling pathway and its potential role in airway remodeling, we measured the expression of Wnt5a/β-Catenin signaling pathway in PM2.5-exposed lung tissues of mice by Western Blot. Compared with the control group, increased expression of Wnt5a and β-Catenin protein was detected in PM2.5 exposed group (Fig 3A-3C), and PM2.5 exposure also signi cantly upregulated PDGFRβ and Tenascin C protein expression in lung tissue of mice (Fig 3D-3F), indicating that PM2.5 exposure induced the expression of Wnt5a/β-Catenin signaling pathway in vivo.

PM2.5 increased Wnt5a/β-Catenin pathway activation in HBSMC
To further investigate the effect of PM2.5 on the expression of Wnt5a/β-Catenin signaling pathway in vitro, we measured the expression of Wnt5a/β-Catenin signaling pathway in PM2.5-exposed HBSMC by Western Blot. Compared with the control group, increased expression of Wnt5a and β-Catenin protein was detected in PM2.5 exposed HBSMC (Fig 4A-4C), and PM2.5 exposure signi cantly upregulated PDGFRβ and Tenascin C protein expression in HBSMC (Fig 4D-4F). We also found increased expression of Wnt5a and β-Catenin at the transcriptional level, as well as downstream target genes TGF-β1, CyclinD1, and cmyc (Fig 4H). Cellular immuno uorescence staining of HBSMC revealed that the expression of β-Catenin was increased after PM2.5 stimulation and intranuclear staining was increased compared to the control group (Fig 4G), which suggested that PM2.5 induced the expression of Wnt5a and activated β-Catenin signaling in HBSMC.
Wnt5a-speci c antagonist BOX5 alleviated PM2.5-induced airway remodeling in mice To investigate the role of Wnt5a in PM2.5 exposure-induced airway remodeling in mice. BOX5, a Wnt5aderived antagonistic peptide was used to block endogenous Wnt5a signaling, then the morphological changes of lung tissues were observed by H & E staining and the expression of α-SMA protein was detected by immunohistochemistry. As depicted in Figure 5A-5E, compared with the PM2.5 group, the airway wall thickness of PM2.5+BOX5-exposed mice thinned, the expression of α-SMA protein in smooth muscle layer decreased, and the ratio of smooth muscle layer to airway thickness (WA%) decreased ( Fig  5A-5E), indicated that BOX5 inhibited PM2.5-induced airway smooth muscle layer thickening and airway remodeling. Moreover, the expression of Wnt5a/β-Catenin signaling pathway in lung tissues of mice were observed by Western Blot. Compared with the PM2.5 group, the expression of Wnt5a, β-Catenin, PDGFRβ and Tenascin C protein was downregulated in the BOX5 + PM2.5 group (Fig 5F-5K), indicated that BOX5 inhibited PM2.5-induced the expression of Wnt5a/β-Catenin signaling pathway during the process of airway remodeling in lung tissues of mice.

5-induced HBSMC proliferation
First of all, the cytotoxicity of BOX5 in HBSMC was examined. HBSMC were stimulated with different doses of BOX5 (100-300 μM) for 48 hours. No signi cantly changes in cell activity were observed at 100 μM( Fig 6D), therefore, 100 μM of BOX5 was subsequently selected as the experimental dose in combination with literature review. As expected, compared with the PM2.5 group, the levels of PCNA and α-SMA showed down-regulation in the BOX5 + PM2.5 group (Fig 6A-6C), indicating that BOX5 inhibited the PM2.5-induced HBSMC proliferation. In addition, BOX5 treatment down-regulated the PM2.5-induced increase of Wnt5a, β-Catenin PDGFRβ and Tenascin C compared to PM2.5 group (Fig 6E-6J). These results suggest that Wnt5a/β-Catenin signaling pathway may participate in the regulation of PM2.5induced HBSMC proliferation in vitro.

Discussion
With the continuous advancement of industrialization and urbanization, PM2.5 is playing an increasingly important role in the development of COPD [20]. Recent epidemiological studies both in China and abroad have found that PM2.5 in air pollution can increase the incidence, risk of acute exacerbation, lung function decline and mortality of COPD [21]. It was demonstrated that the incidence of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) and acute respiratory infections (ARI) is signi cantly increased when PM2.5 pollution is severe [22].
Our ndings demonstrated that PM2.5 exposure caused aberrant upregulation of Wnt5a/β-Catenin signaling pathway both in HBSMC and lung tissue of mice.
Upregulated Wnt5a led to activation of β-Catenin, then increased the expression of TenascinC and PDGFRβ, induced HBSMC proliferation and airway remodeling, targeting the Wnt5a/β-Catenin signaling pathway may serve as an effective therapeutic strategy for PM2.5-induced airway remodeling in COPD.
Epidemiological studies have shown that exposure to PM2.5 poses a serious threat and risk to human health and is signi cantly associated with increased mortality from respiratory disease, lung cancer and cardiovascular disease [23][24][25]. We constructed a mice model by tracheal drip injection of PM2.5. It has been found that COPD-like models can be successfully constructed by tracheal drip injection of PM2.5, which mainly exhibits small bronchial mucosal damage, wall thickening, cell squamous hyperplasia, brous tissue hyperplasia, and brous tissue hyperplasia. Differently from CSE model, PM2.5 mainly causes small airway lesions, such as bronchial dilatation and wall thickening, while CSE mainly shows severe emphysema lesions and poor diffusion function [26][27][28]. Our study also found that PM2.5 exposure led to thickening of the airway wall in mice, especially the smooth muscle layer, and in vitro PM2.5 stimulation of HBSMC led to proliferation, hypertrophy, and upregulation of intracellular proliferation-related protein PCNA expression. Our results suggest that PM2.5 exposure induced emphysema and airway remodeling in mice and induce airway smooth muscle cell proliferation leading to airway remodeling in vitro, which contributes to the development of COPD.
Furthermore, we found that the expression of Wnt5a and β-Catenin increased in HBSMC after PM2.5 exposure, and the expression of TGF-β1, CyclinD1 and C-myc mRNA was upregulated in HBSMC. TGF-β1 is secreted by airway epithelial cells, airway smooth muscle cells and eosinophils, which promotes matrix protein production, contractile protein expression and proliferation of airway smooth muscle cells and airway broblasts [29]. Cyclin D1 and c-myc are core transcription factors and target genes of the Wnt/βcatenin signaling pathway [30]. C-myc and CyclinD1 are widely involved in cell and tissue development and are key downstream effectors of cell proliferation [31,32]. In addition, Wnt5a and β-catenin expression were upregulated in lung tissues of mice exposed to PM2.5. Dysregulation of Wnt5a signaling has been observed in many lung diseases, including COPD, idiopathic pulmonary brosis (IPF) and asthma. Fibroblast-derived Wnt5a has been found to be increased in expression in COPD models and COPD patient specimens [14]. Combined with previous reports, the relationship between Wnt5a/β-catenin signaling pathway and PM2.5-associated airway remodeling was also established in our study.
PDGFRβ is a cell surface tyrosine kinase receptor, transduces extracellular signals to intercellular regions, which promotes cell proliferation, survival and migration [15]. Tenascin C is a large hexameric, multimodal extracellular matrix protein and binds to cell surface receptors, ECM proteins, soluble factors or pathogens to regulate cell adhesion, migration, proliferation and differentiation [33,34]. In a chronic asthma model, blocking the Wnt/β-catenin signaling pathway inhibits airway remodeling, such as subepithelial brosis and smooth muscle proliferation, by downregulating TGF-β and tenascin C/PDGFRβ expression [35]. In accordance with previous studies, we also detected that PM2.5 induced the expression of PDGFRβ and Tenascin C protein in the lung tissue and in HBSMC. Therefore, we speculate that Wnt5a/ β-Catenin signaling pathway is probably involved in PM2.5-induced airway remodeling through regulating tenascin C/PDGFRβ pathway.
To further investigate the mechanisms by which Wnt5a/β-Catenin signaling pathway may involve in PM2.5-associated airway remodeling in COPD, we applied Wnt5a-speci c antagonist BOX5 to block endogenous Wnt5a signaling in mice and HBSMC. Our current data showed that BOX5 do not change the basal expression of Wnt5a, while BOX5 alleviated PM2.5-induced airway wall thickening, increased smooth muscle layer thickness of mice and attenuated PM2.5-induced HBSMC proliferation. It was found that Wnt, TenascinC and PDGFR expression was increased in adult smooth muscle-related lung disease [36].β-Catenin siRNA led to a signi cant decrease in PDGFRβ expression. Moreover, study showed that Wnt signaling regulated PDGFRβ in part by directly regulating Tnc transcription to promote SMC precursor proliferation and adult lung disease development [16]. This study de nes the Wnt/Tnc/PDGFRβ signaling axis, which is essential for lung smooth muscle development and disease progression, as well as nding that this signaling axis is upregulated in both mouse and human models of asthma and pulmonary arterial hypertension. Consistently, we also found that BOX5 decreased β-Catenin activation, and downregulated Tenascin C and PDGFRβ protein expression both in vivo and in vitro, demonstrating that Wnt5a/β-Catenin signaling pathway is predominantly participated in airway remodeling caused by PM2.5 in COPD.

Conclusions
In summary, we investigated the possible role of PM2.5 in COPD by focusing on the airway remodeling mechanism, and demonstrated that PM2.5 induced the upregulation of Wnt5a that in turn promoted β-Catenin activation, increased the expression of Tenascin C and PDGFRβ, which promoted HBSMC proliferation, contributing to airway remodeling in vivo and in vitro. These results provided evidence that Wnt5a/β-Catenin signaling may be used as a new therapeutic approach for PM2.5-associated airway remodeling in COPD.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.