TEW-7197 | Acat Signal

Novel oral transforming growth factor‐β signaling inhibitor potently inhibits postsurgical adhesion band formation

1Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 2Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 3Metabolic Syndrome Research Center, Mashhad University of Medical Sciences, Mashhad, Iran4Department of Modern Sciences and Technologies, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
5Immunology Research Center, Inflammation and Inflammatory Diseases Division, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
6Brighton & Sussex Medical School, Division of Medical Education, Falmer, Brighton, Sussex, UK
7Division of Pulmonary and Critical Care Medicine, Washington University, School of Medicine, Saint Louis, Missouri
8Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
9Department of Laboratory Hematology and Blood Banking, Cancer Molecular Pathology Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 10Orthopedic Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract
Here, we have investigated the therapeutic potency of EW‐7197, a transforming growth factor‐β type I receptor kinase inhibitor, against postsurgical adhesion band formation. Our results showed that this pharmacological inhibitor prevented the frequency and the stability of adhesion bands in mice model. We have also shown that downregulation of proinflammatory cytokines, reduce submucosal edema, attenuation of proinflammatory cell infiltration, inhibition of oxidative stress, decrease in excessive collagen deposition, and suppression of profibrotic genes atthe site of surgery are some of the mechanisms by which EW‐7197 elicits its protective responses against adhesion band formation. These results clearly suggest that EW‐7197 has novel therapeutic properties against postsurgical adhesion band formation with clinically translational potential of inhibiting key pathological responses of inflammation and fibrosis in postsurgery patients.

KEYW ORD S
EW‐7197, fibrosis, inflammation, postsurgical adhesion band formation, TGF‐β signaling
Correspondence
Majid Khazei, MD, Ph.D., Department of Medical Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad 13944‐91388, Iran.
Email: [email protected]

Seyed Mahdi Hassanian, Ph.D., Department of Clinical Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad 13944‐91388, Iran.
Email: [email protected]

Funding information
National Institute for Medical Research Development, Grant/Award Number: 971285; the Biotechnology Development Council of the Islamic Republic of Iran, Grant/Award Number: 970306

*Atena Soleimani and Fereshteh Asgharzadeh contributed equally to this study.
J Cell Physiol. 2019;1–9. wileyonlinelibrary.com/journal/jcp © 2019 Wiley Periodicals, Inc. | 1
SOLEIMANI ET AL.

1 | INTRODUCTION

Intra‐abdominal adhesions are an inevitable pathological condition induced by chemical peritonitis, continuous ambulatory peritoneal dialysis, endometriosis, irradiation, and foreign body reaction. However the majority of adhesion formation is caused by surgical procedures (Hellebrekers & Kooistra, 2011). Adhesion bands are developed in 93% of patients undergoing a peritoneal or gynecolo- gical surgery. The formation of adhesion band is frequently asymptomatic (Brochhausen et al., 2012). However, the condition is potentially correlated with various morbidities and longer operating times due to adhesiolysis (Braun & Diamond, 2014), These pathological adhesions cause postsurgical complications including pelvic pain, female infertility, bowel obstruction and abscess formation (Cai et al., 2018; Ellis et al., 1999). It has been shown that administration of physical barriers with commercial names such as Interceed, Seprafilm, and Adept in damaged sites blocks the linkage between tissues and organs. Using these agents needs to predict where the adhesion bands may form, which results in a limitation in solid barriers application (Cai et al., 2018). Thus, understanding the cellular and molecular mechanisms involved in adhesions formation contribute to the therapeutic strategies.

The balance between cell growth and fibrinolysis is essential in physiologic conditions. Factors impairing this balance can initiate the fibrin clot formation, leading fibrosis (Hellebrekers et al., 2005; Hellebrekers, Trimbos‐Kemper, Trimbos, Emeis, & Kooistra, 2000; Holmdahl, Eriksson, Eriksson, & Risberg, 1998). Trauma and damage to the abdominal wall increase the release of inflammatory cytokines leading to the migration of granulocytes, lymphocytes, and fibroblast to the injured site. Fibroblast‐induced proﬁbrotic molecule trans- forming growth factor (TGF)‐β is highly presented in the injured peritoneum. This cytokine orchestrated a cascade involved in several physiopathological processes. Aberrant regulation of TGF‐β can promote fibrosis and inflammation, induces the synthesis of collagen I and III, and contributes to the generation of extracellular matrix (ECM) proteins (Branford, Klass, Grobbelaar, & Rolfe, 2014).

There are several studies showing that inhibition of the TGF‐β signaling pathway, by using pharmacological inhibitors or neutralizing TGF‐β antibodies, can regulate fibrinogenesis in different cellular and animal models, supporting the therapeutic potency of targeting this signaling pathway in fibrotic‐associated complications (Pennison & Pasche, 2007). EW‐7197 is an oral TGF‐β type I receptor kinase inhibitor with potent antifibrotic characteristics in liver, kidney, and lungs via suppressing TGF‐β/Smad and reactive oxygen species (ROS) signaling (Park et al., 2015). Following oral administration, EW‐7197 is well absorbed and elicits low toxicity in animal models (Jin et al., 2014; Son et al., 2014). In this study, we investigated the therapeutic potential of EW‐7197 in preventing postsurgical peritoneal adhesion band formation, for a better understanding and hence a better management of this disorder. Our results showed that EW‐7197 significantly attenuates adhesion band formation by inhibiting inflammation, oxidative stress, downregulation of proinflammatory genes as well as suppression of fibrosis and profibrotic molecules.

2 | MATERIAL AND METHODS

2.1 | Materials
EW‐7197 was purchased from Cayman Chemical Company (Ann Arbor, MI). Interleukin‐6 (IL‐6), interferon‐γ (IFN‐γ), tissue necrosis factor‐α (TNF‐α) and transforming growth factor‐β (TGF‐β) ELISA kits
were obtained from eBioscience (San Diego, CA). Haematoxylin and eosin, and materials need for malondialdehyde (MDA), total thiol, superoxide dismutase (SOD) and catalase were all purchased from Sigma Co (Saint Louis, MO).

2.2 | Animal experiment
Animal experiments were carried out in accordance with the guideline for Care and Use of Laboratory Animals from Mashhad
University of Medical Sciences (MUMS). BALB/c mice (weighing 30–35 g) were purchased from Pasteur Institute (Tehran, Iran) and maintained according to the protocol approved by Institutional
Animal Care Guidelines. The animals had free access to food and water ad libitum in a temperature (22–25°C) and light (12‐hr light/
dark cycle)‐controlled room.

2.3 | Surgical method
Mice were randomly divided into three groups: (a) A sham group with no adhesion model; (b) A control group with surgical abrasion and peritoneal adhesion; (c) A peritoneal adhesion group treated with
EW‐7197 (2.5 mg·kg−1·day−1; oral gavage; Park et al., 2015) for
7 days. Following anesthesia, adhesion bands were induced by cecal abrasion in peritoneal cavities, which provides a high incidence (100%) of surgical adhesions in control groups after 7 days (Hemadeh, Chilukuri, Bonet, Hussein, & Chaudry, 1993). At the end of the experiments, mice were killed and specimens were rapidly frozen in liquid nitrogen or stored in 10% formalin for further investigations. All animal procedures were carried out according to the guidelines approved by the Ethics Committee of Mashhad University of Medical Sciences.

2.4 | Evaluation of adhesion scores
At the end of the experiment procedure, the peritoneal cavity was opened via a U‐shaped incision. Grading of the adhesion bands was performed in a blinded fashion, using the Nair (Nair, Bhat, & Aurora,
1974) and Leach (Leach, Burns, Dawe, SmithBarbour, & Diamond, 1998) adhesion scoring system for evaluating the frequency and stability of the adhesion bands, respectively.

2.5 | Histological staining
Tissue sample were fixed in formalin, and embedded in paraffin. Tissues were sectioned using a microtome, and stained with haematoxylin and eosin (H&E) as well as Masson’s trichrome. H&E staining was performed to indicate the inflammatory cells infiltration, whereas Masson staining was used to investigate the collagen deposition, representing the fibrosis score.

2.6 | Enzyme‐linked immunosorbent assay (ELISA)
Commercially available ELISA kits were used to measure the levels of proinflammatory cytokines including IL‐6, IFNγ, TNF‐α, and TGF‐β in tissue homogenates as described previously (Dinarvand, Hassanian,
Weiler, & Rezaie, 2015).

2.7 | Real‐time polymerase chain reaction (RT‐PCR) Total RNA from tissues samples were isolated using the RNeasy Mini kit purchased from Qiagen Inc. (Hilden, Germany). Complementary DNAs
(cDNAs) were synthesized from 1 μg of extracted RNA by a cDNA Reverse Transcription Kit according to the manufacturer’s protocol
(Takara Bio, Shiga, Japan). For real‐time quantitative RT‐PCR, equivalent
amounts of synthesized cDNAs were subjected to the Ampliqon SYBR Green PCR Master Mix. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) used as housekeeping control gene to normalize the
messenger RNA (mRNA) expression levels. The expressions of fibrotic and inflammatory genes were evaluated using gene‐specific primer pairs (Table 1) obtained from Macrogene Co. (Seoul, Korea).

2.8 | Evaluation of oxidative stress markers
The oxidative stress was assessed via measuring the level of malondialdehyde (MDA), nitrite and total thiol as well as the catalase and superoxide dismutase (SOD) activities in the tissue homogenates as described previously (Asgharzadeh et al., 2017; Hashemzehi et al., 2018; Marjaneh et al., 2018).

2.9 | Cell culture
The 3T3 cell line was obtained from the American Type Culture
Collection (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat‐ inactivated fetal bovine serum (FBS) and 1% streptomycin/penicillin.
Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C.

2.10 | Reactive oxygen species (ROS) detection
Intracellular ROS production was measured using 2′,7′‐dichlorodihy- drofluorescein diacetate (DCFDA) cellular reactive oxygen species (ROS) detection assay kit according to the manufacturer’s instruc- tions (Abcam, Cambridge, MA). Accordingly, 3T3 cells (5 × 103 cells/ well) were seeded under standard conditions in a 96‐well culture plate. DCFDA solution (100 μl) directly added to culture medium for
30 min. In the following step, cells were treated with lipopolysac- charide (LPS; 5 µg/ml; Song, Kim, Yoon, & Kim, 2006) alone and LPS combined with EW‐7197 (1 µM). A fluorescence plate reader

2.11 | Quantification of apoptosis
Apoptosis analysis was performed using Annexin V‐FITC (fluorescein isothiocyanate) detection assay kit (Cayman Chemical) (Giovannetti et al., 2014). Cells were treated with EW‐7197 (1 µM) for 24 hr. Following centrifugation, 200 μl 1× Annexin binding buffer was
added, centrifuged and then the cells were resuspended in Annexin V‐FITC/PI (propidium iodide) solution. After 15 min incubation in room temperature, the viable, necrotic and apoptotic cells were
quantified by BD FACSCalibur (BD Biosciences) flow cytometer, following the manufacturer’s instruction.

2.12 | Western blot analysis
Analysis of western blot analysis accomplished as described (Hassanian, Dinarvand, Smith, & Rezaie, 2015). Following treatment, Total protein was extracted by lysis buffer and protein concentration was measured using pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL). Protein samples were loaded equally, separated by sodium dodecyl sulphate polyacryla-
mide gel electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon‐P, Millipore, Bedford, MA). Followed by blocking and incubating with primary and secondary
antibodies, Signals were visualized using a West Femto Chemilumi- nescent reagent (Thermo Scientific, Rockford, IL).

2.13 | Statistical analysis
Results were expressed as mean values ± SEM. All differences were considered to be statistically significant at a p < .05. Statistical
comparisons were determined using t test and the Wilcoxon Mann‐
Whitney tests for parametric and nonparametric data, respectively.

3 | RESULTS

3.1 | EW‐7197 decreases incidence and rigidity of postsurgical adhesion bands formation
All animals survived to 7 days after surgery and no significant differences were found in body weight in three groups. During the
EW‐7197 decreases the frequency and the stability of adhesion bands. (a) A Schematic representation of the experimental protocol. (b) Treatment of mice (six mice in each group) with EW‐7197 (2.5 mg/kg) significantly decreased adhesion formations in peritoneum postsurgery, compared with the untreated group. (c, d) EW‐7197 reduces the adhesion bands frequency (c) and stability (d) in EW‐7197 treated mice, compared with the control group. *p < .05 [Color figure can be viewed at wileyonlinelibrary.com] EW‐7197 attenuates inflammation in adhesion mice model. (a–c) Hematoxylin and eosin (H&E) staining in the postoperative peritoneal adhesion tissues represented a significant morphological damage to abdominal tissues in untreated adhesion group, compared with EW‐7197‐treated one. Arrows indicate inflammatory cells infiltration. (d–f) The effects of EW‐7197 on concentrations of inflammatory cytokines; (d) interleukin‐6 (IL‐6), (e) interferon‐γ (IFN‐γ) and (f) tumor necrosis factor‐α (TNF‐α) was compared between groups. (g,h) EW‐7197 decreased inflammation by reducing the protein concentration of TNF‐α (g) and phosphorylated P‐65 subunit (h) in 3T3 fibroblast cells. The assay was performed in triplicates. *p < .05 [Color figure can be viewed at wileyonlinelibrary.com] study, no significant complications including bleeding, infection or delay in wound healing was observed in EW‐treated group before the second laparotomy. Schematic representation of the experimental protocol is presented in Figure 1a. Macroscopic analysis indicated that EW‐7197 prevents the formation of intra‐abdominal adhesion bands in EW‐ treated mice, compared with the untreated group (Figure 1b). For quantitative evaluation of the EW‐7197 effects on frequency and stability of adhesion bands, we used two Nair (Nair et al., 1974) and Leach (Leach et al., 1998) scoring systems. Scoring system represented a
significant reduction in incidence and rigidity of adhesion bands in EW‐ treated mice (Figure 1c,d). Data analysis presented that EW‐7197‐ treated adhesion model had a significant lower Nair and Leach score than the control group (Figure 1c,d) (p < .05). No adhesion band was found near the peritoneal incision in the sham group.

3.2 | EW‐7197 attenuates inflammation in adhesion mice model
To determine the protective effect of EW‐7197 on adhesion band symptoms, we evaluated inflammation as a key pathological response during peritoneal adhesion band formation, between groups. Results of H&E staining revealed that EW‐7197 decreased the inflammatory cell infiltration and morphological damage to abdominal tissues (Figure 2a,c). Next, we compared the effect of EW‐7197 on the tissue concentrations of inflammatory cytokines including IL‐6, TNF‐α and IFNγ between EW‐7197‐treated and untreated mice. We showed that EW‐7197 significantly reduced proinflammatory cytokine levels, compared with the control group (Figure 2d–f). Consistent with in vivo results, western blot analysis showed that EW‐7197 treatment decreased the inflammation via reduction of TNF‐α (Figure 2g), phosphorylation o P‐65 subunit of nuclear factor κB (NF‐κB; Figure 2h) in 3T3 cell line. These results along with the inhibitory effects of EW‐7197 on inflammatory cell infiltration suggested the potent anti‐inflammatory properties of this specific TGF β type I receptor kinase inhibitor in postsurgical adhesion formation.

3.3 | EW‐7197 inhibits inflammation by suppressing oxidative stress postsurgery
To further investigate anti‐inflammatory mechanisms by which EW‐ 7197 attenuates abdominal adhesions, we compared the oxidant/
antioxidant balance between different groups. Our results showed that EW‐7197 treatment reduced levels of oxidative markers including nitrite and MDA in tissue homogenates, compared with the positive control adhesion mice (p < .05; Figure 3a,b). Consistently, measuring levels of antioxidant markers showed that the total thiol concentration, as well as catalase and superoxide dismutase (SOD) activities, were elevated in EW‐7197‐treated mice, in comparison to control group, supporting the antioxidant effect of EW in adhesion bands (p < .05; Figure 3c–e). Moreover, the analysis of ROS detection
presented that EW‐7197 is able to decrease LPS‐induced ROS accumulation in 3T3 cell line (Figure 3f). These results suggest that

Antioxidant responses of EW‐7197 in adhesion bands. The concentrations of (a) nitrite, (b) MDA, (c) total thiol, (d) SOD activity, and (e) catalase activity were measured in the absence and presence of EW‐7197 in tissue homogenates of adhesion mice model. Oxidative responses were reduced in EW‐197 group, compared with untreated adhesion band group. The assay was performed in triplicates. (f) EW‐7197 decreased the LPS‐ induced ROS generation in 3T3 cell line. Assay conducted in three independent experiments. *p < .05; **p < .01; and ***p < .001. MDA, malondialdehyde; LPS, lipopolysaccharides; ROS, reactive oxygen species; SOD, total thiol, superoxide dismutase [Color figure can be viewed at wileyonlinelibrary.com]

EW‐7197 treatment induced apoptosis in fibroblast cells. (a) Both early and late apoptosis were increased via EW‐7197 treatment in 3T3 cell line. (b) EW‐7197 induced the sub G1 population, compared with control. (c, d) the regulatory effect of EW‐7197 on proapoptotic P‐53 (c) and BAX (d) mRNAs was quantified and compared between groups. All values presented are performed in triplicate. *p < .05 and **p < .01. BAX, BCL2 associated X; mRNA, messenger RNA EW‐7197 exerts its protective effects, at least partially, by regulating the oxidant/antioxidant balance, alleviating oxidative stress reactions in adhesion bands formation.

3.4 | EW‐7197 promoted apoptotic cell death in standard fibroblast 3T3 cell line
To determine whether EW‐7197 induced apoptosis, we assessed the fractional DNA content of EW‐7197‐treated fibroblastic 3T3 cells, using Annexin V‐FITC/PI staining. Apoptosis analysis revealed that EW‐7197 increased both early and late apoptotic cell death. Furthermore, induction of sub G1 population was shown in the presence of EW‐7197 treatment (Figure 4a,b). Consistently, we evaluated the regulatory effect of EW‐7197 on proapoptotic proteins, tumor suppressor P‐53 (Figure 4c), apoptotic regulator BCL2 associated X (BAX) (Figure 4d), at mRNA levels in 3T3 cells.
Our results showed that the expression of P‐53 and BAX mRNAs were increased in EW‐7197‐treated fibroblast cells, suggesting the regulatory role of EW‐7197 on cell‐cycle progression and cellular apoptosis.

3.5 | EW‐7197 suppresses fibrosis in intraperitoneal adhesion bands
We used Masson’s trichrome staining to compare the collagen deposition in peritoneal tissues between groups. Consistent with anti‐inflammatory role of EW‐7197, administration of EW‐7197 suppressed collagen deposition and fibrosis compared with the positive control (untreated) group as visualized by Masson's trichrome staining (Figure 5a–c). Due to the critical role of TGF‐β in regulating fibrosis, (Schnaper, Hayashida, Hubchak, & Poncelet, 2003) we measured protein concen- trations of this molecule in peritoneal tissues. Our results showed that EW‐7197 potently reduced the TGF‐β concentration compared with the nontreated group (Figure 5d). We further evaluated the relative mRNA expression of two fibrogenic factors including collagen 1a1 (Col 1a1) and Col 1a2. Results showed that mRNA levels of Col 1a1 and Col 1a2 were significantly decreased in EW‐7197‐treated group, compared with the untreated adhesion group (Figure 5e,f). Taken together, these results supported the antifibrotic properties of EW‐7197 against fibrogenic‐associated complications in postsurgical adhesion bands.

EW‐7197 inhibits fibrosis in adhesion mice model. (a–c) Fibrosis and collagen deposition were assessed by Masson’s trichrome staining. lamina propria (*), skeletal muscle (#), and fibrotic tissue (↑) are marked in the (d) The effects of EW‐7197 on concentrations of TGF‐β protein was compared between groups. (e, f) EW‐7197 significantly decreased the mRNA level of fibrotic factors including Col 1a1 and Col 1a2 compared with the untreated group. Experiments performed in triplicate. **p < .01 and ***p < .001. mRNA, messenger RNA; TGF‐β, transforming growth factor‐β [Color figure can be viewed at wileyonlinelibrary.com]

4 | DISCUSSION

We investigated the potential antifibrotic and anti‐inflammatory responses of TGF‐β type1 receptor kinase inhibitor, EW‐7197, in the postsurgical intra‐abdominal adhesion bands in an experimental mice model. Here, we show that EW‐7197 reduced TGF‐β tissue levels,potentially attenuated
fibrogenesis and inhibited collagen deposition. Further studies showed that EW‐7197 could prevent adhesion formation by attenuating inflammation, reducing inflammatory cell infiltration, decreasing concentrations of proinflammatory cytokines, and inhibiting the oxidative stress. These results strongly supported the therapeutic potential of EW‐7197 in preventing postoperative adhesion band formation.

Cellular and animal experiments have shown that EW‐7197 downregulates the expression of profibrotic genes (collagen, fibronectin, integrin, and α‐SMA), decreased ROS formation, and suppressed ECM accumulation by blocking TGF‐β/Smad2/3 signal- ing in fibrotic model of liver, kidney and lung (Park et al., 2015). In line with these results, Kim et al. (2016) exhibited that oral administration of EW‐7197 could inhibit cholestatic liver fibrosis through inhibition of hypoxia‐inducible factors‐1α (HIF‐1α)‐in- duced TGF‐β signaling activation in a rat model. Consistent with these findings, our findings showed that this inhibitor significantly attenuated profibrotic‐associated complications in postsurgical adhesion band formation in mice model, supporting the therapeutic potential of EW‐7197 in treatment of fibrosis‐related adhesion bands.

Furthermore, there are studies showing that injury‐induced inflammation enhances postoperative peritoneal adhesions forma- tion (Chegini, 2008; Rout, Saed, & Diamond, 2002). Moreover, surgery‐induced inﬂammation increases macrophage recruitment and fibroblasts infiltration to the injured site, enhancing adhesion formation in peritoneum (Koh & DiPietro, 2011). Due to the complexity of the regulatory role of TGF‐β in inflammation, administration of EW‐7197 can lead to diverse and even contra- dictory results. In line with this, administration of EW‐7197 after
stent placement in rat esophagus, only reduced the level of collagen deposition and thickness of submucosal fibrosis. However, there was no effect on the infiltration of inflammatory cells during wound healing process (Jun et al., 2017). Similarly, Han et al. (2018) showed that EW‐7197‐coated metallic stents did not modify inflammatory cell infiltration after stent placement in the urethras of tested dogs.

In contrast to these results, we recently showed that EW‐7197 elicits protective responses against ulcerative colitis‐associated inflamma- tory responses by reducing submucosal edema, inflammatory cell infiltration, and downregulating of proinflammatory genes in in-flamed and fibrotic ulcerative colitis tissues (Binabaj et al., 2018). The role of TGF‐β signaling pathway in postsurgical adhesion band is not completely understood. Moreover, the precise molecular mechan- isms of the protective role of EW‐7197 are still required further cellular and animal and preclinical studies in postsurgical adhesion band formation patients.

ACKNOWLEDGMENTS

This study was supported by grants awarded by the Biotechnology Development Council of the Islamic Republic of Iran (Grant No. 970306) and National Institute for Medical Research Development (Grant No. 971285).

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

AUTHOR CONTRIBUTIONS

A. S., F. A., and F. R. performed cellular and animal experiments. A. S. and S. M. H. with support from G. A. F. and M. R wrote the manuscript. J. N. carried out in silico analysis and performed the computations. S. M. measured ELISA experiments. M. F., M. H. A., and M, M, B. contributed to cellular and animal studies. A. R. A. provided key reagents. M. R. P., and S. H. S. analyzed data and contributed to the interpretation of the results. S. M. H., A. A., and M. K. designed the study plan and supervised the project. All authors discussed the results and contributed to the final manuscript.

DATA ACCESSIBILITY
Research data are not shared.

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