Experimental Lung Research

ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20

RhoA/Rho-kinase activation promotes lung fibrosis in an animal model of systemic sclerosis Yihua Bei, Thong Hua-Huy, Carole Nicco, Sy Duong-Quy, Nhat-Nam Le-Dong, Kiet-Phong Tiev, Christiane Chéreau, Frédéric Batteux & Anh Tuan DinhXuan To cite this article: Yihua Bei, Thong Hua-Huy, Carole Nicco, Sy Duong-Quy, Nhat-Nam LeDong, Kiet-Phong Tiev, Christiane Chéreau, Frédéric Batteux & Anh Tuan Dinh-Xuan (2016) RhoA/Rho-kinase activation promotes lung fibrosis in an animal model of systemic sclerosis, Experimental Lung Research, 42:1, 44-55, DOI: 10.3109/01902148.2016.1141263 To link to this article: http://dx.doi.org/10.3109/01902148.2016.1141263

Published online: 12 Feb 2016.

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Date: 15 February 2016, At: 03:42

EXPERIMENTAL LUNG RESEARCH , VOL. , NO. , – http://dx.doi.org/./..

ORIGINAL ARTICLE

RhoA/Rho-kinase activation promotes lung fibrosis in an animal model of systemic sclerosis Yihua Beia,b,∗ , Thong Hua-Huya,∗ , Carole Niccoc , Sy Duong-Quya , Nhat-Nam Le-Donga,d , Kiet-Phong Tieva,e , Christiane Chéreaub , Frédéric Batteuxb , and Anh Tuan Dinh-Xuana a

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Laboratoire de Physiologie Respiratoire, Université Paris Descartes, Sorbonne Paris Cité, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France; b Regeneration Lab and Experimental Center of Life Sciences, School of Life Science, Shanghai University, Shanghai, China; c Laboratoire d’Immunologie Clinique, Universite Paris Descartes, Sorbonne Paris Cite, Equipe Batteux, Institut Cochin, Hopital Cochin, Assistance Publique-Hopitaux de Paris (AP-HP), Paris, France; d Department of Pneumology, St. Elisabeth Hospital, Namur, Belgium; e Department of Internal Medicine, Hospital of Vitry sur Seine, Site Pasteur, Vitry sur Seine, France

ABSTRACT

ARTICLE HISTORY

Background: Systemic sclerosis (SSc) is a connective-tissue disease characterized by vascular injury, immune-system disorders, and excessive fibrosis of the skin and multiple internal organs. Recent reports found that RhoA/Rho-kinase (ROCK) pathway is implicated in various fibrogenic diseases. Intradermal injection of hypochlorous acid (HOCl)-generating solution induced inflammation, autoimmune activation, and fibrosis, mimicking the cutaneous diffuse form of SSc in humans. Our study aimed firstly to describe pulmonary inflammation and fibrosis induced by HOCl in mice, and secondly to determine whether fasudil, a selective inhibitor of ROCK, could prevent lung and skin fibroses in HOCl-injected mice. Methods: Female C57BL/6 mice received daily intradermal injection of hypochlorous acid (HOCl) for 6 weeks to induce SSc, with and without daily treatment with fasudil (30 mg·kg−1 ·day−1 ) by oral gavage. Results: HOCl intoxication induced significant lung inflammation (macrophages and neutrophils infiltration), and fibrosis. These modifications were prevented by fasudil treatment. Simultaneously, HOCl enhanced ROCK activity in lung and skin tissues. Inhibition of ROCK reduced skin fibrosis, expression of α-smooth-muscle actin and 3-nitrotyrosine, as well as the activity of ROCK in the fibrotic skin of HOCl-treated mice, through inhibition of phosphorylation of Smad2/3 and ERK1/2. Fasudil significantly decreased the serum levels of anti-DNA-topoisomerase1 antibodies in mice with HOCl-induced SSc. Conclusions: Our findings confirm HOCl-induced pulmonary inflammation and fibrosis in mice, and provide further evidence for a key role of RhoA/ROCK pathway in several pathological processes of experimental SSc. Fasudil could be a promising therapeutic approach for the treatment of SSc.

Received  June  Accepted  January 

Introduction Systemic sclerosis (SSc) is a heterogeneous connectivetissue disease characterized by vascular injury, immune-system disorders, and excessive fibrosis of the skin and a variety of internal organs.[1] Pulmonary complications, including pulmonary fibrosis and pulmonary hypertension, represent two principal causes of death in patients with SSc.[2] Anatomopathological analyses of lung autopsies from patients with SSc evidenced local inflammatory foci intercalated with fibrotic lesions, supporting the hypothesis that pulmonary inflammation precedes and promotes lung fibrosis.[3] Increasing evidence also suggests

KEYWORDS

fasudil; pulmonary inflammation; pulmonary fibrosis; Rho-kinase; systemic sclerosis

that pulmonary endothelial cells activation also paves the way to fibrotic lesions in various interstitial lung diseases,[4] including SSc,[3, 5] and idiopathic pulmonary fibrosis.[5] Our previous report showed that active alveolitis could predict pulmonary function deterioration or mortality in patients with SSc.[6] Recent observations suggest the crucial role of oxidative stress in human SSc.[7–9] Mice treated with hypochlorous acid (HOCl)-generating solution showed autoimmune activation, inflammation and skin fibrosis, mimicking the diffuse cutaneous form of SSc in humans.[10] However, pulmonary lesions including leukocytes infiltration, fibroblasts activation,

CONTACT Anh Tuan Dinh-Xuan [email protected] Service de Physiologie-Explorations Fonctionnelles, Hôpital Cochin,  Rue du Faubourg Saint-Jacques,  Paris, France. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ielu. ∗ Yihua Bey and Thong Hua-Huy contributed equally to this manuscript. ©  Taylor & Francis

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and collagen overproduction have not yet been well described in this animal model of SSc. The small GTPase RhoA and its effectors, Rhokinases (ROCK), play an essential role in the development of pulmonary hypertension by causing and worsening endothelial dysfunction, vasoconstriction, and vascular remodeling.[11, 12] Recent studies have shown that RhoA/ROCK is implicated in several fibrogenic diseases and that fasudil, a selective inhibitor of ROCK, reduced inflammatory and fibrotic changes.[13–16] It has been recently demonstrated that ROCK stimulates myofibroblast differentiation and overproduction of extracellular matrix in SSc fibroblasts in vitro, and that TGF-β activates ERK in a ROCK-dependent manner.[17] In the present study, we used the mouse model of SSc induced by intradermal injection of HOCl and we tested the preventive effects of fasudil, a specific inhibitor of ROCK, on HOCl-induced pulmonary and skin injuries in order to assess the role of the RhoA/ROCK pathway information of skin and lung fibrogenesis in this animal model.

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USA) and this or a vehicle was administrated by gavage 5 days per week for 6 weeks. Three days after the period of injections was completed, mice were intraperitoneally anesthetized with ketamine (100 mg/kg) and xylazine (15 mg/kg), the thorax was opened and the lungs were sampled after blood removal by right ventricular puncture. Skin biopsies were obtained from the injection sites with a punch (6 mm diameter). Left lungs were perfused with 10% formalin for histopathological analyses. Right lungs and one part of injected skin samples were stored at −80°C until experiments for collagen content or protein extraction. Dermal thickness

The dermal thickness within the injection sites was measured with a caliper and expressed in millimeters (mm). Measurements were performed before the first injection and then once a week until the day of sacrifice, by the same operator. Collagen contents in skin and lung

Materials and methods Animal handling and experimental protocol

Six-week-old female C57BL/6 mice were purchased from Harlan (France). All mice were housed in autoclaved ventilated cages with a 12-hour day–night cycle and free access to water and food according to the animal guidelines for biomedical research applied in our institution. Mice were randomly divided into three groups: (i) phosphate-buffered saline (PBS) control (n = 5); (ii) HOCl (n = 6); (iii) HOCl+fasudil (n = 6). Daily intradermal injections of substances that generated HOCl were used to induce SSc in mice, as previously described.[10] HOCl was prepared by adding a NaClO solution (9.6% active chlorine) to a KH2 PO4 solution (100 mM, pH 6.2) just before the daily injection. HOCl concentration was determined by measuring the optical density at 280 nm, adjusted to an optical density of between 0.7 and 0.9. A total of 500 μL of HOCl was injected intradermally into two posterior sites in mice (250 μL per site) for 5 days per week, for 6 weeks (HOCl-mice). The control group received daily injections of 500 μL of PBS (PBS-mice). Treatment with fasudil (30 mg·kg−1 ·day−1 ) was dissolved in PBS at a dilution of 2 mg/mL (LC Laboratories, Woburn, MA,

Skin biopsies from injected sites and right-lung tissues were diced into small pieces and then mixed with pepsin (1:10 weight ratio) and 0.5 M acetic acid at room temperature for one week. Collagen contents in skin and lung were determined by the quantitative dyebinding SircolTM Collagen Assay (Biocolor Ltd, Carrickfergus, UK), as previously described.[10] Results were presented as μg of collagen/mg of skin (or lung) tissue. Lung histopathological analyses

Left lungs were embedded in paraffin wax. A series of 5 μm-thick sections were prepared from the midportions of the paraffin-embedded tissues, and stained with hematoxylin-eosin (Sigma-Aldrich) for routine examination, and were stained with picro-sirius red (Sigma-Aldrich) for lung-fibrosis analysis. Slides were examined with a light microscope at a 100× magnification (Leica DC300, Leica Microsystems, Wetzlar, Germany). Immunohistochemical staining

To evaluate the profiles of lung inflammatory cells, macrophages, neutrophils, T lymphocytes (CD3),

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and B lymphocytes (CD20) were immunohistochemically stained with macrophage-marker antibodies (1:100, Santa Cruz, CA, USA, sc-101447), neutrophilmarker antibodies (1:1000, Santa Cruz, CA, USA, sc-71674), CD3 antibodies (1:400, Santa Cruz, CA, USA, sc-101442), and CD20 antibodies (1:250, Santa Cruz, CA, USA, sc-7735), respectively, as previously described.[16] For analyses of each inflammatory cell, approximately 20–25 fields of each lung section were randomly chosen and microscopically photographed (×200). The number of lung interstitial inflammatory cells in each photographed field was counted by two blinded observers. Results were presented as the number of each inflammatory cell type per photographed field (×200), and calculated from the mean of 5–6 animals per group. Immunohistochemical staining of the same lung sections was also performed with anti-iNOS antibodies (1:100, Santa Cruz, CA, USA, sc-651). The main steps were performed, as described above, except that antigen retrieval for iNOS was done with sodium citrate buffer (10 mM, pH 6.0) heated in a microwave for 20 min. For analyses, 20 fields of each lung section were microscopically photographed (×200) and analyzed by two blinded observers. Expression of epithelial iNOS was semi-quantitatively scored according to the following scale: (0) no staining; (1) focal staining; (2) diffuse weak staining; (3) diffuse moderate staining; and (4) diffuse strong staining. The mean of scores for every study animal was used for statistical analyses.

Immunoblotting

Western blotting procedures were performed as previously described.[16] Briefly, skin biopsies and lung samples, were homogenized in complete RIPA lysis buffer. The primary and secondary antibodies used for western blotting are presented in the Table 1. Immunoreactivities were revealed using enhanced chemiluminescence (ECL) prime Western Blotting detection reagent (RPN2232, Amersham). Densitometric analysis was performed using ImageJ software. Membranes were then stripped and re-blotted for β-actin, ERK1/2, and Smad2/3, as appropriate. Sample volumes were normalized by β-actin. Phosphorylation of ERK1/2 and Smad2/3 was normalized by total ERK1/2 and Smad2/3, respectively.

Table . Characteristics of primary and secondary antibodies used for western blotting. Target molecule Primary antibodies Nitrotyrosine α-SMA p-ERK/ TGF-β p-Smad/ ERK/ Smad/ β-actin Secondary antibodies Anti-rabbit Anti-goat Anti-mouse

Species isotype Mouse IgGb Rabbit IgG Goat IgG Rabbit IgG Goat IgG Goat IgG Goat IgG Mouse IgG

Reference (clone)

Dilution

sc- (HM-) : sc- (G-) : sc- (Thr /Tyr ) : sc- (V) : sc- (Ser /) : sc- (MK) : sc- (I-) : sc- (C) :,

Goat IgG-HRP Rabbit IgG-HRP Goat IgG-HRP

sc- sc- sc-

:, :, :,

∗ Anti-ICAM- antibody was purchased from R&D Systems. All other antibodies

were from Santa Cruz Biotechnologies.

Measurement of ROCK activity

ROCK activity was quantified by MYPT1 phosphorylation on Thr-696, relative to total MYPT1 expression, by western blot analyses of protein extracts from lung and skin tissues. The western-blot procedure was performed, as previously described,[16] using rabbit antiphospho-MYPT1 (Thr-696) antibody (1:500, Upstate, ABS45) as primary antibody, and goat anti-rabbit IgGHRP (1:5,000, Santa Cruz, CA, USA, sc-2004) as secondary antibody. Membranes were then stripped and re-blotted for MYPT1 (1:1000, Santa Cruz, CA, USA, sc-25618). ROCK activity was presented as the ratio of p-MYPT1 (Thr-696) to total MYPT1 protein-band density. Serum levels of Anti-DNA-topoisomerase-1 antibody

Serum levels of anti-DNA topoisomerase-1 IgG antibody were measured by ELISA using purified calf thymus DNA topoisomerase-1 coated on the wells of a 96well polystyrene microplate. Plates were blocked with PBS-1% BSA, and then 100 μL of 1:50 mouse serum was added in duplicate and allowed to react for 1 h at room temperature. Bound antibodies were detected with alkaline phosphatase-conjugated goat anti-mouse IgG antibodies, and the reaction was developed by adding p-nitrophenyl phosphate. Optical density was measured at 450 nm and the optical density in blank wells was subtracted.[10] Statistical analyses

All analyses were performed using SPSS (Version 20.0) software. Data are expressed as mean ± SEM.

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Figure . Fasudil reduced infiltration of macrophages and neutrophils in the lung of HOCl-mice. (A) Numbers of infiltrated macrophages per field (-fold magnification) in lung sections immunohistochemically stained. (B) Numbers of infiltrated neutrophils per field (-fold magnification) in lung sections immunohistochemically stained. (C) Representative images of immunohistochemical staining of Fasudil group). ∗ P < ., ∗∗ P < ., NS = not significant. lung sections for macrophages and neutrophils (n =  in PBS group, n =  in HOCl or HOCl + Fasudil group).

Comparisons were performed with one-way analysis of variance among multiple groups, followed by Tukey’s post hoc tests as appropriate. A P-value of < .05 was considered statistically significant. Figures are presented using GraphPad Prism 5 software. Results Pulmonary infiltration of macrophages and neutrophils induced by HOCl was prevented by fasudil

We first aimed to verify whether intradermal injections of HOCl generating solution could induce pulmonary inflammation. Local inflammation was evaluated by

immunohistochemical staining for specific markers of principal leukocytes including macrophages, neutrophils, and lymphocytes. There was a significant increase in lung interstitial infiltration of macrophages (5.8 fold; P < .01) (Figure 1A) and neutrophils (1.8 fold; P < .05) (Figure 1B) in HOCl-mice as compared with PBS-mice. Increased numbers of lung leukocytes infiltration were completely prevented by fasudil treatment (P < .01 for macrophages, and P < .05 for neutrophils, respectively). Representative images of lung sections stained for macrophages and neutrophils are presented in Figure 1C. No significant difference was found in lung infiltration of lymphocytes T or B in HOCl-mice as compared with PBS-mice (data not shown).

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Figure . Fasudil reduced inducible nitric oxide synthase (iNOS) expression in the lung of HOCl-mice. (A) Semi-quantitative scoring for iNOS expression in lung sections as assessed by immunohistochemical stains. (B) Representative images of lung sections immunohistochemically stained for iNOS (-fold magnification). (C) Western blot for iNOS expression in the lung. (n =  in PBS group, n =  in HOCl or HOCl + Fasudil group).∗∗ P < .; ∗∗∗ P < .; NS = not significant.

Increased pulmonary expression of iNOS induced by HOCl was attenuated by fasudil

Immunohistochemical staining showed that the expression of iNOS, a biomarker of inflammation, was significantly higher in the bronchial epithelium of HOCl-mice as compared with PBS-mice (1.4 fold; P < .01). Fasudil reduced iNOS expression in the lungs of HOCl-mice as compared with untreated HOCl-mice (P < .01) (Figure 2A-B). The results were confirmed by Western Blot (Figure 2C).

Fasudil decreased the expression of 3-nitrotyrosine and the high Rho-kinase activity in the lungs of HOClmice. HOCl induced 3-nitrotyrosine (3-NT) expression in the lungs of HOCl-treated mice. Fasudil prevented the augmentation of 3-NT expression as evaluated by Western Blot (Figure 3A).The activity of ROCK was quantified by the relative expression of phosphorylated form of MYPT1 (p-MYPT1) in whole-lung extracts. HOCl injections significantly increased the ROCK

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Figure . HOCl induced -nitrotyrosine (-NT) expression and Rho-kinase activity in the lungs of HOCl-treated mice. Fasudil prevented the augmentation of -NT expression and ROCK activity in lung tissues of HOCl-mice. Expression of -NT was evaluated by Western Blot (A) and ROCK activity was assessed by relative expression of the phosphorylated MYPT (Thr) on total MYPT (B) (n =  in PBS group, n =  in HOCl and HOCl + Fasudil groups).∗ P < .; ∗∗ P < .; ∗∗∗ P < .; NS = not significant.

activity by 1.5 fold as compared with PBS injections (P < .05). Treatment with fasudil efficiently reduced the elevated activity of ROCK in HOCl-mice (P < .05) (Figure 3B). Besides, there was no significant difference in ROCK1/2 expression between lungs taken from the three groups of mice (data not shown). Fasudil reduced lung and skin fibroses in HOCl-intoxicated mice

Intradermal injections of HOCl caused significant lung fibrosis in mice compared with injections of PBS, as measured quantitatively by the dye-binding SircolTM collagen assay (1.33 fold versus PBS; P < .05) (Figure 4A). Fasudil significantly reduced the accumulation of lung collagen in HOCl-mice compared with untreated HOCl-mice (P < .05) (Figure 4A). Histopathological analysis of lung tissues stained with picro-sirius red evidenced the reduction of lung collagen deposition in mice treated with fasudil (Figure 4B). Western blotting was performed for α-SMA expression and the relative phosphorylation of ERK1/2 in the lung extracts. Fasudil prevented the increased expression of α-SMA in the lung (Figure 4C) whereas there was no significant modification of p-ERK1/2 on total ERK1/2 expression (P < .05) (Figure 4D). In the injured skin, HOCl induced a significantly greater dermal thickness compared with injections of PBS (2.4 fold; P < .01) (Figure 4E). The collagen content in skin biopsies was significantly increased in HOCl-mice versus mice treated with PBS (1.8 fold; P < .01) (Figure 4F). Fasudil significantly reduced both dermal thickness (P < 0.01) (Figure 4E) and collagen content (P < .01) (Figure 4F) in HOCl-mice when compared to untreated HOCl-mice.

Fasudil reduced the expression of 3-nitrotyrosine and attenuated the increased activity of ROCK in the skin of HOCl-mice

Expression of 3-NT in injured skin, used as a marker of nitrosative stress in vivo, was markedly higher in HOCl-mice (4.5 fold; P < .05) compared to PBS-mice, as measured by immunoblotting. 3-NT expression was significantly attenuated by treatment with fasudil (P < .05) (Figure 5A). We performed western blotting on proteins extracted from mouse skin to investigate whether the RhoA/ROCK pathway was activated at the directly injured tissue. The relative expression of p-MYPT1/total MYPT1 in the skin of HOCl-mice was significantly higher (1.6 fold; P < .001) than that in PBS-mice. Fasudil significantly reduced the high expression of p-MYPT1/MYPT1 in the skin samples from HOCl-mice (P < .001) (Figure 5B). Fasudil reduced α-SMA expression and phosphorylation of Smad2/3 and ERK1/2 in the skin of HOCl-mice

Expression of α-SMA in the fibrotic skin of HOCl-mice was higher than that from PBS-mice (1.8 fold; P < .05), which was significantly attenuated by treatment with fasudil (P < .05) (Figure 6A). There was a significant increase in TGF-β1 expression in the skin of HOClmice (2.5 fold; P < .05) as compared with that in PBSmice. However, fasudil treatment did not significantly inhibit the elevated expression of TGF-β1 in the skin of HOCl-mice (P > .05) (Figure 6B). Greater amounts of p-Smad2/3 (1.9 fold; P < .05) and pERK1/2 (3.6 fold; P < .05) were found in the skin of HOCl-mice versus

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Figure . Effects of fasudil on lung and skin fibrosis in HOCl-mice. (A) Collagen contents in lung, as measured by dye-binding sircol method. (B) Representative images showing collagen deposition in lung tissues stained with picro-sirius red (-fold magnification). (C) Western blot for α-SMA expression in the lung. (D) Western blot showing relative phosphorylation of ERK/ in the lung (E) Dermal thickness, measured with a caliper in the injection sites in mice. (F) Collagen contents in skin, as measured by dye-binding sircol method. (n =  in PBS group, n =  in HOCl or HOCl + Fasudil group).∗ P < ., ∗∗ P < ., NS = not significant.

PBS-mice (Figure 6C-D). The increased phosphorylation of Smad2/3 and ERK1/2 in the skin of HOCl-mice was significantly reduced by treatment with fasudil (P < .05 for both cases) (Figure 6C-D).

Fasudil decreased the serum levels of anti-DNAtopoisomerase-1 antibodies in HOCl-mice

Serum levels of anti-DNA topoisomerase-1 antibodies were significantly increased in HOCl-mice compared

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Figure . HOCl induced -nitrotyrosine (A) and Rho-kinase activity (B) in the skin of HOCl-treated mice. Fasudil prevented the augmentation of -NT and ROCK activity in the skin of HOCl-mice. Expression of -NT was evaluated by immunoblotting. ROCK activity was assessed by relative expression of the phosphorylated MYPT (Thr) on total MYPT (n =  in PBS group, n =  in HOCl and HOCl + Fasudil groups).∗ P < .; ∗∗∗ P < .; NS = not significant.

to those in PBS-mice (1.9 ± 0.26 arbitrary unit (AU) versus 0.48 ± 0.03 AU, with P < .05), equivalent to a 4fold increase in anti-Scl-70 antibodies (Figure 7). Treatment with fasudil significantly reduced the serum levels of anti-DNA topoisomerase-1 antibodies in HOClmice (0.73 ± 0.17 AU vs. 1.9 ± 0.26 AU, P < .05) (Figure 7). Discussion Recent observations have demonstrated the implication of the RhoA/ROCK pathway as evidenced by the inhibitory effect of fasudil and Y-27632, two selective inhibitors of ROCK on inflammatory and fibrotic changes in various fibrotic experimental diseases.[13–16] In this study, we found increased activity of ROCK in lung and skin tissues from HOCl-mice, and treatment with fasudil prevented both lung and skin fibrosis in SSc-induced mice. Our results also showed an inhibitory effect of fasudil on HOCl-induced increased expression of α-SMA without affecting expression of ERK1/2. Implication of the ROCK signaling pathway in pulmonary lesions induced by intradermal injection of HOCl is further supported by inhibitory effects of fasudil on infiltration of macrophages and neutrophils in the lungs. Pulmonary fibrosis and pulmonary hypertension represent two main causes of death in patients suffering from SSc.[2] In the present study, HOCl generated pulmonary fibrosis as previously described.[10, 18]

However, the pathophysiological mechanism underlying HOCl-induced pulmonary fibrosis has not yet been well identified. In these reports, increased lung interstitial infiltration of neutrophils and macrophages,[18] associated with high expression of iNOS in bronchial epithelium, was found in HOCl-mice. In the present study, we further show that the increased infiltration of neutrophils and macrophages, and the increased iNOS expression in lungs, was significantly reduced by fasudil in HOCl-mice. Inflammatory cells are implicated in the production of various pro-inflammatory and pro-fibrotic cytokines and chemokines, which lead to activation of fibroblasts, inducing their migration, proliferation, and differentiation into myofibroblasts, responsible for pulmonary fibrosis.[3,19] Increased numbers of inflammatory cells and mediators in the bronchoalveolar lavage fluid taken from SSc patients with interstitial lung disease were associated with ongoing inflammation and progressive pulmonary fibrosis.[20, 21] In addition, the recruitment of inflammatory cells into the lungs, as well as the secretion of cytokines and chemokines, are responsible for inducing iNOS expression and for augmenting pulmonary production of NO, which is suggested to be a biomarker for the presence of alveolar inflammation, even before the occurrence of lung fibrosis in SSc.[22, 23] Thus, the reduction of lung fibrosis in fasudil-treated mice might be related to decreased lung inflammation. Excessive production of reactive oxygen species (ROS) plays a critical role in the pathogenesis of

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Figure . Fasudil reduced the expression of α-SMA and inhibited the phosphorylation of Smad/ and ERK/ in the skin of HOCl-mice. (A) Western blot for α-SMA expression in the skin. (B) Western blot for TGF-β expression in the skin. (C) Western blot showing relative phosphorylation of Smad/ in the skin. (D) Western blot showing relative phosphorylation of ERK/ in the skin (n =  in PBS group, n =  in HOCl or HOCl + Fasudil group). ∗ P < ., NS = not significant.

SSc,[3, 7–9, 24, 25] and ROCK can be activated by ROS production,[26] An auto-amplification loop linking ROS, Ras, and ERK1/2 has been shown to enhance cell responses to growth factors, and leads to DNA damage and collagen synthesis in SSc skin fibroblasts.[27] Intradermal HOCl is supposed to stimulate ROS production in local fibroblasts, leading to Ras protein activation and ERK1/2 phosphorylation.[28] In the present study, we found an increased phosphorylation of ERK1/2 in the fibrotic skin of HOCl-mice and demonstrated that fasudil significantly reduced ERK1/2 phosphorylation in the skin of HOCl-mice, linking RhoA/ROCK activation with ERK1/2 phosphorylation in the pathogenesis

of skin fibrosis. Recent reports have shown that TGFβ 1 activates ERK1/2 in a ROCK-dependent manner, and that ERK1/2 phosphorylation mediates the stimulatory effects of ROCK on myofibroblast differentiation and extracellular matrix synthesis in SSc fibroblasts.[17] Thus, reduced phosphorylation of ERK1/2 in fibrotic skin might account for the beneficial effect of fasudil against dermal fibrosis in HOCl-mice. In addition, results of the present study are consistent with previous reports using the same animal model and showing that cutaneous and lung fibrosis induced by HOCl can be prevented by simvastatin[29] and propylthiouracil[30] through their inhibitory effects on ROS production,

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Figure . Fasudil decreased the serum levels of anti-DNAtopoisomerase- antibody in HOCl-injected mice. The serum levels of anti-DNA-topoisomerase- antibody in the  groups of mice were measured by ELISA method (n =  in PBS group, n =  in HOCl or HOCl + Fasudil group). ∗ P < ., NS = not significant.

further supporting the role of oxidative stress, and reinforcing the importance of RAS family-specific therapy in SSc. Besides ROS, excessive reactive nitrogen species are also implicated in the pathogenesis of SSc. Nitrotyrosine (3-NT), formed in the presence of NO and excessive ROS, is used as a biomarker of inflammation and nitrosative stress in SSc.[31, 32] High levels of 3-NT in diseased tissues are considered to be responsible for vascular injury and fibrogenic reaction.[18, 33] In our study, the increased expression of 3-NT in the fibrotic skin of HOCl-mice was significantly reduced by treatment with fasudil, which partly explains the therapeutic effect of fasudil in skin fibrosis in experimental SSc. In addition, the elevated expression and activity of ROCK were significantly inhibited by fasudil in the fibrotic skin of HOCl-mice, suggesting the important role of the RhoA/ROCK pathway in inflammation and oxidative stress. Previous reports have shown that the RhoA/ROCK pathway enhances inflammation by inducing pro-inflammatory molecules, such as IL6 and monocyte chemoattractant protein-1.[34, 35] This pathway also induces overexpression of endothelial NADPH oxidase and increases angiotensin II-triggered ROS production.[36] The role of RhoA/ROCK in nitrosative stress induction and the mechanism underlying the anti-nitrosative effect of fasudil in the diseased skin of HOCl-mice needs to be further studied. Previous studies have demonstrated abnormal activation of immune cells, characterized by the

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high production of autoantibodies and inflammatory mediators in HOCl-mice, which links local and systemic fibrosis.[28] In the present study, serum levels of anti-DNA topoisomerase-1 antibodies were significantly increased in HOCl-mice compared with those in PBS-mice. Fasudil, administrated by daily oral gavage, reduced the production of autoantibodies in HOCl-mice, implying the role of the RhoA/ROCK pathway in immune-system activation. The underlying mechanism remains to be further investigated. In summary, our results confirmed the presence of pulmonary inflammation and fibrosis in the murine model of HOCl-induced SSc and demonstrated that selective inhibition of ROCK prevents skin fibrosis and lung fibrosis in this model. Reduction of skin fibrosis by fasudil occurred through inhibition of phosphorylation of Smad2/3 and ERK1/2 in TGF-β 1 signaling, whilst attenuation of lung fibrosis was probably caused by the decreased pulmonary inflammation. The RhoA/ROCK pathway could be a novel therapeutic target for the treatment of SSc.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Funding This study was supported by grants from the Legs Poix, University of Paris, the Air Liquide Foundation, and the Program of International Science & Technology Cooperation (Grant Number: 2012DFG31440) from the Ministry of Science and Technology, People’s Republic of China.

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Rho-kinase activation promotes lung fibrosis in an animal model of systemic sclerosis.

Systemic sclerosis (SSc) is a connective-tissue disease characterized by vascular injury, immune-system disorders, and excessive fibrosis of the skin ...
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