Long-term treatment with fasudil improves bleomycin-induced pulmonary fibrosis and pulmonary hypertension via inhibition of Smad2/3 phosphorylation Yihua Bei a, b , Thông Hua-Huy a , Sy Duong-Quy a , Viet-Ha Nguyen a , Weihua Chen a, b , Carole Nicco c , Frédéric Batteux c , Anh Tuan Dinh-Xuan a, b, * a Paris Descartes University, Medical School, Assistance Publique Hôpitaux de Paris, Service de Physiologie, EA 2511, Hôpital Cochin, 27 Rue du Faubourg Saint-Jacques, 75014 Paris, France b Clinical and Translational Research Center, Tongji University School of Medicine and Shanghai East Hospital,150 Jimo Road, Shanghai 200120, China c Paris Descartes University, Medical School, Assistance Publique Hôpitaux de Paris, Laboratoire d’Immunologie, EA 1833, Hôpital Cochin, 27 Rue du Faubourg Saint-Jacques, 75014 Paris, France article info Article history: Received 24 May 2013 Received in revised form 26 July 2013 Accepted 27 July 2013 Keywords: RhoA Rho-kinases Bleomycin Pulmonary hypertension Pulmonary fibrosis abstract Pulmonary hypertension (PH) associated with pulmonary fibrosis (PF) considerably worsens prognosis of interstitial lung diseases (ILD). RhoA/Rho-kinases (ROCK) pathway is implicated in high pulmonary vascular tone and pulmonary fibrosis but the effect of ROCK inhibitors on PH associated with PF is not known. We therefore aimed to determine whether long-term treatment with fasudil, a selective ROCK inhibitor, could attenuate PF and PH induced by bleomycin in mice. Male C57BL/6 mice received a single dose of intratracheal bleomycin (3.3 U/kg) to induce PF. Treatment with fasudil (30 mg kg 1 day 1 ) was given intraperitoneally for 7, 14 or 21 days until mice underwent hemodynamic measurements. Right ventricular systolic pressure (RVSP) and RV/(LV þ S) ratio were assessed. Lung inflammatory cells pro- files, including macrophages, neutrophils, lymphocytes B and lymphocytes T were assessed by immu- nohistochemistry. Lung fibrosis was evaluated by histological and biochemical methods. Pulmonary arteriole muscularization and medial wall thickness (MWT) were evaluated by immunohistochemical staining for a-SMA. Bleomycin induced severe PF and PH in mice, associated with an increased RhoA/ ROCK activity in the lung. Fasudil reduced lung inflammation and lung collagen content, and attenuated the increased RVSP, RV hypertrophy, and pulmonary vascular remodeling inbleomycin-intoxicated mice. Fasudil inhibited the increased activity of RhoA/ROCK pathway, and partly altered bleomycin-associated activation of TGF-b1/Smad pathway, via inhibition of Smad2/3 phosphorylation. The efficacy of long- term treatment with fasudil suggests that the blockade of RhoA/ROCK pathway may be a promising therapy for patients with ILD-associated PH. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Pulmonary hypertension (PH) associated with pulmonary fibrosis (PF) considerably worsens prognosis of chronic interstitial lung diseases (ILDs). The most common ILDs causing PF and PH include idiopathic pulmonary fibrosis (IPF), connective tissue dis- orders (CTDs) and sarcoidosis. To date, no specific treatment for PH associated with PF is available. On the other hand, high-quality clinical trials investigating current conventional therapies such as prostacyclins, endothelin-1 (ET-1) receptor antagonists and phos- phodiesterase type 5 (PDE5) inhibitors are scarce in patients with ILD-associated PH [1], thus highlighting the need for finding new therapies for these patients. There is increasing evidence to suggest that the RhoA/Rho- kinase (ROCK) pathway is involved in various vascular disorders in the systemic and the pulmonary circulations [2]. RhoA, one of the best-known members of Rho GTPases, and its effector proteins, ROCKs, are strongly implicated in a wide range of cell functions, Abbreviations: PH, pulmonary hypertension; PF, pulmonary fibrosis; ILD, inter- stitial lung disease; IPF, idiopathic pulmonary fibrosis; CTD, connective tissue dis- ease; ROCK, Rho-kinase; BALF, bronchoalveolar lavage fluid; PVR, pulmonary vascular resistance; RVSP, right ventricular systolic pressure; MWT, medial wall thickness; BLM, bleomycin. * Corresponding author. Service de Physiologie e Explorations Fonctionnelles, Hôpital Cochin, Université Paris Descartes, 27 Rue du Faubourg Saint-Jacques, 75014 Paris, France. Tel.: þ33 158412341; fax: þ33 158412345. E-mail address: anh-tuan.dinh-xuan@cch.aphp.fr (A.T. Dinh-Xuan). Contents lists available at ScienceDirect Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt 1094-5539/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pupt.2013.07.008 Pulmonary Pharmacology & Therapeutics xxx (2013) 1e9 Please cite this article in press as: Bei Y, et al., Long-term treatment with fasudil improves bleomycin-induced pulmonary fibrosis and pulmonary hypertension via inhibition of Smad2/3 phosphorylation, Pulmonary Pharmacology & Therapeutics (2013), http://dx.doi.org/ 10.1016/j.pupt.2013.07.008