Role of Rho-kinase and its inhibitors in pulmonary hypertension

Pharmacology & Therapeutics 137 (2013) 352–364

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Associate Editor: M.G. Belvisi

Role of Rho-kinase and its inhibitors in pulmonary hypertension Sy Duong-Quy a, Yihua Bei a, b, Zhongmin Liu b, Anh Tuan Dinh-Xuan a, b,⁎ a Paris Descartes University, Medical School, Assistance Publique Hôpitaux de Paris, Service de Physiologie, Explorations Fonctionnelles. 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

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Keywords: RhoA Rho-kinase Pulmonary hypertension

a b s t r a c t Pulmonary hypertension (PH) is an incurable disease with a dreadful survival rate. The disease is characterized by sustained vasoconstriction, progressive vascular remodeling, and irreversible right heart dysfunction. While hypoxic pulmonary vasoconstriction (HPV) is known to be the main pathophysiological factor causing the rise in pulmonary arterial pressure, biological mechanisms leading to HPV and vascular remodeling are multiple and complex and, as yet, incompletely understood. It is thought that molecular interactions and cross talks are involved in the pathogenesis of PH, perturbing the physiological balance between substances controlling vascular tone, cell growth and apoptosis. This balance is achieved by subtle interactions between factors acting as both vasodilators and inhibitors of cell growth like nitric oxide, prostacyclin, vasoactive intestinal peptide and molecules with potent vasoconstrictor and cell growth activities like endothelin-1. Recent in vivo studies showed that the Rho GTPase/RhoA pathway and its downstream effectors, the Rho-kinases (ROCK-1 and ROCK-2), had an important role in PH, due to its lasting effects on vasoconstriction and pulmonary cell proliferation leading to vascular remodeling. Beneficial effects obtained in vivo with Rho-kinase inhibitors (e.g.Y-27632 and fasudil) in experimental PH will hopefully lead to future clinical trials with new compounds selectively targeting this pathway, which is now proven to be detrimental when over-activated in both experimental animals and human patients. © 2012 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . 2. Overview of pulmonary hypertension . . . . . . . . . . . . 3. Rho-kinase signaling pathway . . . . . . . . . . . . . . . . 4. Vascular downstream effect of Rho-kinase . . . . . . . . . . 5. Role of Rho-kinase signaling in pulmonary hypertension . . . . 6. Rho-kinase inhibitors in the treatment of pulmonary hypertension 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: 5-HT, serotonin; AA, arachidonic acid; AC, adenylate cyclase; Ang II, angiotensin II; ATP, adenosine triphosphate; BMP, bone morphogenetic proteins; CaM, calmodulin; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CH, chronic hypoxia; COPD, chronic obstructive pulmonary disease; EC, endothelial cell; ECE, endothelin converting enzyme; EGF, epidermal growth factor; eNOS or NOS-3, endothelial nitric oxide synthase; ERK, extracellular signal-regulated kinases; ET-1, endothelin-1; GDP, guanosine diphosphate; GTP, guanosine triphosphate; MAPK, mitogen-activated protein kinases; MCT, monocrotaline; MLC, myosin light chain; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; NE, norepinephrin; NO, nitric oxide; PAH, pulmonary arterial hypertension; PAP, pulmonary arterial pressure; PDGF, platelet-derived growth factor; PH, Pleckstrin-homology; PH, pulmonary hypertension; PIP2, phosphatidyl-inositol-diphosphate; PKC, protein kinase C; PLC, phospholipase C; PVR, pulmonary vascular resistance; RBD, Rho-binding domain; Rho-GAP, Rho GTPase activating protein; Rho-GEF, Rho guanine nucleotide exchange factor; ROCK, Rho-kinases; ROS, reactive oxygen species; RV, right ventricle; RVSP, right ventricle systolic pressure; SERT, serotonin transporter; sGC, soluble guanylate cyclase; SMC, smooth muscle cell; SPC, sphingosine phosphorylcholine; TXA2, thromboxane A2; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Service de Physiologie, Explorations Fonctionnelles, Hôpital Cochin Hospital, Université Paris Descartes, 27, rue du faubourg Saint-Jacques, 75104 Paris, France. Tel.: +33 158412341; fax: +33 158412345. E-mail address: [email protected] (A.T. Dinh-Xuan). 0163-7258/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pharmthera.2012.12.003

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1. Introduction Pulmonary hypertension (PH) is a rare disorder with a dreadful prognosis. Despite substantial advances in the knowledge of pathogenesis and therapy during the last decade, PH still remains an incurable disease. In the 90s, the median survival rate of untreated patients was around 2.8 years from diagnosis (D'Alonzo et al., 1991). PH is defined by an increase in mean pulmonary arterial pressure (PAP) ≥25 mm Hg at rest, a pulmonary wedge pressure (PWP) ≤15 mm Hg and a normal or reduced cardiac output, assessed by right heart catheterization (Galiè et al., 2009). PH is currently classified into five groups and one subgroup according to pathological, clinical, and therapeutic features (Galiè et al., 2009). Current treatment of PH consists of the use of conventional therapy in combination with specific treatments with continuous prostacyclin infusion or inhalation, oral phosphodiesterase-5 inhibitors (Olschewski et al., 2002; Sitbon et al., 2002; Sastry et al., 2004) and oral endothelin-1 receptor antagonists (Channick et al., 2001; Rubin et al., 2002; Naeije & Huez, 2007). None of these drugs, however, can be considered as an optimal treatment for PH as they mainly act as vasodilators and lack inhibitory effects on remodeling of the pulmonary vasculature. There is therefore a need for new treatments to be found and tested by randomized clinical trials (Hoeper & Dinh-Xuan, 2004). Recently, accumulating evidence showed that RhoA and its downstream effectors, the Rho-kinases, have a preponderant role in the physiopathology of PH due to their potent effects on pulmonary arterial smooth muscle cell (SMCs) contraction and proliferation (Nagaoka et al., 2006; Homma et al., 2008; Guilluy et al., 2009). This paper will review the biological role of Rho-kinase signaling pathway and the efficacy of Rho-kinase inhibitors in the treatment of PH, targeting both its vasoconstrictor and vascular remodeling components. 2. Overview of pulmonary hypertension 2.1. Classification of pulmonary hypertension The first international classification of PH, established in 1973 at the World Health Organization (WHO) Symposium held in Evian, categorized the disorder as “primary” when no underlying etiology or risk factor could be identified, and “secondary” when clearly associated to a clearly identified primary disorder (Hatano & Strasser, 1975). Since then, the clinical classification of PH has been revised thrice in 1998 (Evian, France), 2003 (Venice, Italy) (Simonneau et al., 2004), and 2008 (Dana Point, USA) (Galiè et al., 2009). In these classifications, clinical conditions with PH were divided into five groups according to pathological, pathophysiological, and therapeutic characteristics. The latest clinical classification, made in Dana Point in 2008 (Galiè et al., 2009) maintains the overall structure of the EvianVenice classifications while amending some specific points to avoid possible confusion among the terms PH and PAH (pulmonary arterial hypertension). This new classification of PH is summarized as follows: Group 1 (PAH): including idiopathic PAH, heritable PAH (germline mutations of BMPR2, ALK1 or endoglin genes), drugs and toxins induced PAH, PAH associated with other conditions (connective tissue diseases, HIV infection, portal hypertension, congenital heart disease, schistosomiasis, chronic hemolytic anemia), and persistent pulmonary hypertension of the newborn. Group1′: PH due to pulmonary veno-occlusive disease and/or pulmonary capillary hemangiomatosis. These conditions have been classified as a distinct category but not completely separated from PAH, and designated as Group 1′. Group 2: PH related to left heart disease, including systolic dysfunction, diastolic dysfunction, and valvular disorders.

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Group 3: PH related to lung disease and/or hypoxia, including chronic obstructive pulmonary disease, interstitial lung disease, other pulmonary disease with mixed restrictive and obstructive patterns, sleep-disordered breathing, alveolar hypoventilation disorders, chronic exposure to high altitude, and developmental abnormalities. Group 4: PH due to chronic thromboembolic disease (CTEPH: chronic thromboembolic pulmonary hypertension) without precise criterion to distinguish between proximal and distal forms. Group 5: PH with unclear and/or multifactorial mechanisms, including heterogeneous conditions with different pathological features such as hematological disorders (myeloproliferative disorders, splenectomy), systemic disorders (sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, vasculitis), metabolic disorders (glycogen storage disease, Gaucher disease, thyroid disorders), and others (tumoral obstruction, fibrosing mediastinitis, chronic renal failure on dialysis). 2.2. Physiopathology and pathogenesis of pulmonary hypertension Pulmonary vasoconstriction is an important feature of all forms of PH. Decrease of alveolar partial pressure of oxygen results in acute pulmonary vasoconstriction which, when prolonged, will eventually lead to the occurrence of PH (Sommer et al., 2008). Chronic hypoxic pulmonary vasoconstriction is sustained and worsened by an imbalance between vasodilators and vasoconstrictors acting on the pulmonary vascular smooth muscle cells. Recent evidence also suggests deleterious roles of growth factors (Giaid & Saleh, 1995; Tuder et al., 1999; Galié et al., 2004). Other mechanisms involved in the physiopathology of PH include 1/reduced cross-sectional area of pulmonary vascular bed due to disorders affecting the lung parenchyma, 2/thrombi obstructing pulmonary vascular lumen (Mahapatra et al., 2006a, 2006b), 3/overloaded volume and pressure caused by left-right intracardiac shunts, 4/obstruction of pulmonary venous flow, and 5/a combination of the above mechanisms in advanced stage of PH (Ito et al., 2003; Montani et al., 2009). Whilst the classification of PH is well defined and largely depends on clinical features, the pathogenesis of PH is complex and incompletely understood. It is thought that the underlying mechanisms are multiple and lead to both functional and structural changes of the pulmonary vasculature (Humbert et al., 2004; Yuan & Rubin, 2005). 2.3. Pathology and pathobiology of pulmonary hypertension Although different mechanisms are involved in the physiopathology and pathogenesis of the disease, all forms of PH have common pathologic features in pulmonary arteries. It is mostly characterized by vascular remodeling (medial hypertrophy, intimal fibrosis, and adventitial thickening), and organized thrombotic (Pietra et al., 2004; Yuan & Rubin, 2005; Tuder et al., 2009). Pulmonary vascular remodeling is characterized by the increasing of external diameter of pulmonary arteries due to predominantly SMC hypertrophy and extracellular matrix and collagen deposit (Palevsky et al., 1989; Yi et al., 2000). However, the correlation between morphological changes, degree of disease severity, and potential response to vasodilators is not clearly established in PH (Tuder & Zaiman, 2004). The pathobiology of PH is very complex and multifactorial (Humbert et al., 2004; Hassoun et al., 2009; Morrell et al., 2009) (Fig. 1). Many mediator and signaling pathway with its downstream effectors are implicated in the pathobiology of PH. It is involved in the regulation of pulmonary vasoactivity, endothelial function, SMC proliferation, and vascular inflammation or thrombosis. Impairment of vasoconstriction– vasodilatation balance is the earliest disorder occurring in the pathogenesis of PAH (Christman et al., 1992; Giaid & Saleh, 1995). Endothelial

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Fig. 1. Potential mechanisms involved in the development of PH. Top half: Relaxation of smooth muscle cells induced by NO, prostacyclin, and VIP is mediated by cGMP and cAMP whereas constriction induced by TXA2, ET-1 and 5-HT is mediated by membrane receptors coupled to G proteins and intracellular calcium modifiers such as IP3 (inositol triphosphate) or mediators of RhoA/Rho-kinase pathways. Bottom half: Proliferation of smooth muscle cells is induced by cytokines, growth factors and TGF-β. Control of transcription is regulated by the BMP proteins and its membrane receptors via Smad signaling effectors, and MAP kinase pathways. Abbreviations: 5-HT1B/2A, serotonin receptors 1B or 2A; BMPR2, bone morphogenetic protein receptor 2; ETA/B, endothelin receptors A or B; GF, growth factors; GPCR, G protein-coupled receptors; HHV-8, human herpesvirus 8; JNK, c-Jun N-terminal kinase; Kv, potassium voltage-gated channels; P, phosphorylation; PGH2, prostaglandin H2; PGI2, prostaglandine I2 (prostacyclin); PGI2-R, prostacyclin receptors; R-TXA2, thromboxane receptors; VPAC-1/2, vasoactive intestinal peptide receptors 1 or 2; TGF-β, transforming growth factor-β; TPH1, tryptophan hydroxylase 1; VIP, vasoactive intestinal peptide.

dysfunction leads to chronically impaired production of vasodilator and anti-proliferative agents such as NO and prostacyclin, along with overexpression of vasoconstrictor and proliferative substances such as thromboxane A2 and endothelin-1 (ET-1). Inflammatory cells, platelets (through the serotonin pathway), and prothrombotic abnormalities also play a significant role in pathogenesis of PH. However, the role of other molecular and signaling pathway, which is mediated by many factors such as ET-1, serotonin (5-HT), angiotensin II (Ang II), growth factor etc. …, in pathobiology and pathogenesis of PH is not clearly clarified. 2.4. Genetic disorders in pulmonary hypertension The best characterized genetic defects in heritable pulmonary arterial hypertension are mutations of the gene encoding bone morphogenetic protein receptor type 2 (BMPR2), a member of the transforming growth factor-β signaling family. BMPR2 modulates the growth of vascular cells by activating the intracellular pathway of Smad and LIM kinase (Foletta et al., 2003). The germline mutations in BMPR2 gene are detected in at least 70% of cases of

heritable pulmonary arterial hypertension (Machado et al., 2006, 2009). BMPR2 gene mutations are also detected in 11%–40% of apparently sporadic cases, thus representing the major genetic predisposing factor for PAH (Sztrymf et al., 2008). More than 45 different mutations of BMPR2 gene have been identified in patients with heritable PAH (Lane et al., 2000; Newman et al., 2001). Functional studies have shown that point mutations and truncations in the kinase domain exert dominant negative effects on receptor function (Newman et al., 2004), resulting in incomplete penetrance and genetic anticipation. Mutations of other receptors such as activin receptor-like kinase 1 (ALK1) and endoglin have also been identified in PAH patients usually from families with coexistent hereditary hemorrhagic telangiectasia (Trembath et al., 2001). Mutations in ALK1 are believed to result in cellular growth-promoting via Smad-depending signaling. Genetic mutations of the serotonin transporter (5-HTT) are more frequent in idiopathic PAH than control subjects (Eddahibi et al., 2001). The L-allelic variant of the 5HTT gene is associated with an increased expression of the transporter and increased proliferation of vascular SMC. Serotonin gene polymorphism has also been found in PH

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patients with hypoxemic chronic obstructive pulmonary disease (COPD) (Eddahibi et al., 2003).

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3. Rho-kinase signaling pathway 3.1. Molecular structure and expression of Rho-kinase

2.5. Role of inflammation The roles of inflammation and autoimmunity in vascular remodeling seen in idiopathic PH and PH associated with systemic diseases have been recently highlighted (Tuder & Voelkel, 1998; Voelkel et al., 1998). First, circulating autoantibodies directed against endothelial cells and cell nuclei are frequently found in PH (Isern et al., 1992; Dorfmüller et al., 2003; Tamby et al., 2005). Secondly, perivascular infiltration of inflammatory cells including lymphocytes (T and B), macrophages, and dendritic cells, is a constant feature of plexiform vascular lesions (Tuder et al., 1994; Pietra et al., 2004; Perros et al., 2007b). Cytokines and inflammatory chemokines are also involved in the pathogenesis of PH. Increased concentrations and expressions of pro-inflammatory cytokines (IL-1β, IL-6) were found in plasma and lung tissue of patients with severe idiopathic PAH (Humbert et al., 1995). In mice, high expression of IL-6 was associated with increased pulmonary vascular resistance, and extensive pulmonary vascular lesions (Steiner et al., 2009). Efficacy of tocilizumab, a monoclonal antibody directed against IL-6 receptors, has been recently reported in a patient with PH (Furuya et al., 2010). Plasma concentrations and expressions of fractalkine and its receptors (CX3CR1 and CX3CL1) are increased in circulating lymphocytes (CD4+ and CD8+) and lung tissue (Balabanian et al., 2002), and the role of the chemokines CCL5 (RANTES) and CCL2 (chemokine ligand 2) studied in PH patients (Perros et al., 2007a; Sanchez et al., 2007). 2.6. Pulmonary vascular remodeling Pulmonary vascular remodeling is a major pathological feature of PH. It is due to encroach of vascular walls into the lumen, thus reducing the vessels inner diameter. Many cell types of pulmonary vessel and circulation contribute to the development of pulmonary vascular remodeling. This remodeling is characterized by the thickening of all the three layers of pulmonary vessels, including the media, the intima, and the adventitia. It is marked by the hypertrophy and hyperplasia of different cell types such as endothelial cells (EC), smooth muscle cells (SMC), and fibroblasts. These structural changes are enhanced by the accumulation of extracellular matrix components, including collagen, elastin, fibronectin, and tenascin, in the vascular walls, and predominantly in the adventitia (Jeffery & Wanstall, 2001). In the remodeled of pulmonary vessels, the thickened and hypertrophied media layer critically reduces vascular lumen whilst perturbing the fragile balance between vascular contraction and relaxation mediators. Medial hypertrophy results from imbalance between proliferation and apoptosis of SMC, occurring at all levels of pulmonary vascular tree, from large arteries to small arterioles (Dingemans & Wagenvoort, 1978). Furthermore, medial hypertrophy further obstructs vascular lumen through extension of new muscle cells into muscular and non muscular pulmonary arterioles, termed as the muscularization (Wagenvoort & Wagenvoort, 1970). Muscularization of pulmonary vessels is initiated by the differentiation of many precursor cells and fibroblasts into SMC (Jones et al., 1999; Sata, 2006). Pulmonary vascular remodeling is also characterized by hypertrophy of the intima. Lung injury caused by hypoxia, inflammation, and shear stress, results in endothelial cells damage and dysfunction, uncontrolled cell proliferation, excessive vasoconstriction, and in situ thrombosis. These processes are linked to complex patterns of inflammation, angiogenesis, and trans-differentiation of endothelial cells to pulmonary vascular SMC (Zhu et al., 2006; Sakao et al., 2007), leading to intimal proliferation and lamina-intima fibrosis and yielding plexiform lesions of pulmonary arteries.

Rho-kinase, also named Rho-associated kinase and identified in mild 1990s, is one of the effectors of Rho families (Ishizaki et al., 1996). Rho-kinase or ROCKs having two different isoforms, ROCK-1or ROCKβ and ROCK-2 or ROCK-α, is the main downstream effectors of GTPase-RhoA (Ishizaki et al., 1996). ROCKs are serine/threonine kinases with a molecular mass of ~160 kD. These kinases are formed by parallel homodimers including a catalytic (kinase) domain in its amino-terminus (NH2- or N-terminal domain), a coiled-coil in its middle dimerization portion, and a putative Pleckstrin-homology (PH) domain in its cystein-riche domain (COOH- or C-terminal domain, CRD) (Fig. 2) (Ishizaki et al., 1996; Matsui et al., 1996; Fukata et al., 2001). These carboxyl terminal domains constitute an autoinhibitory region that reduces the kinase activity of ROCKs (Amano et al., 1999). The Rho-binding domain (RBD) of ROCKs is localized in the C-terminal portion of the coiled-coil region, and it shows sequence homology to the Rho-interaction domain of kinectin which is a regulating protein of microtubule-based organelle motility (Alberts et al., 1998) (Fig. 2). The coiled-coil region of ROCKs is showed to interact with other α-helical proteins, whereas the PH domain is involved in protein localization (Riento & Ridley, 2003). ROCKs mRNAs are expressed in invertebrates and in vertebrates. In human, ROCK-1 and ROCK-2 are encoded by two different genes localizing on chromosome 18 (18q11.1) and chromosome 2 (2p24), respectively (Nakagawa et al., 1996; Takahashi et al., 1999). Two isoformes of Rho-kinase in human have the homologue structure with about 65% of amino acid and 58% of RBD. The highest similarity (92%) is presented at kinase domain (Leung et al., 1995; Nakagawa et al., 1996). The mRNAs of ROCK-1 and ROCK-2 are ubiquitously expressed, with a preferential expression of ROCK-2 mRNA in brain and skeletal muscle. Both ROCK-1 and ROCK-2 are expressed in vascular smooth muscle and in heart (Leung et al., 1996; Wibberley et al., 2003). Cell-fractionation studies show that ROCKs are mainly distributed in the cytoplasm fraction but a small amount of ROCKs is also found in the membrane fraction (Leung et al., 1996; Matsui et al., 1996). In addition, ROCK-1 might colocalize with centrosomes (Chevrier et al., 2002). However, ROCKs are also found in subcellular localization at the vimentin intermediate-filament network and at actin stress fibers (Sin et al., 1998; Kawabata et al., 2004). 3.2. Regulation of Rho-kinase activity In the structure of ROCKs, the C-terminus (Rho-binding domain and PH domain) plays a role as a dominant-negative autoinhibitor (Fig. 3) due to its independent interaction with the catalytic domain (N-terminus) (Amano et al., 1999). Lacking of the C-terminus of ROCKs (truncated forms) are constitutively formed the active form kinase (Amano et al., 2000). Beside of the self-associative autoinhibition, the activity of ROCKs is also influenced by its affinity for ATP which is regulated by the dimerization of kinases (Doran et al., 2004). Binding of GTPase-RhoA (amino acids 23–40 and 75–119 of RhoA) to ROCKs at Rho-binding domain induces conformational changes of ROCKs, resulting in relieve of autoinhibitory blockage of kinase activity (Fujisawa et al., 1998). This binding is believed to stimulate the phosphotransferase activity of ROCKs (positive regulation) by disrupting the interaction between catalytic and the C-terminal region of proteins (Loirand et al., 2006), which thereby frees the kinase activity (Fig. 4a). Independently of RhoA, ROCK activity might be activated by other stimulators such as arachidonic acid (AA), sphingosine phosphorylcholine (SPC), caspase-3 or granzyme B (Feng et al., 1999b; Shirao et al., 2002; Sebbagh et al., 2001, 2005). AA and SPC interact with the negative regulatory region at PH domain, thus disrupting its inhibitory

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Fig. 2. The molecular structure of ROCKs. The kinase domain is located at the amino terminus (N-terminus) of the protein, followed by coiled-coil region containing the Rho-binding domain (RBD). The Pleckstrin-homology (PH) with an internal cystein-rich domain (CRD) is situated in the carboxyl terminus (C-terminus).

property on the catalytic activity of ROCKs (Feng et al., 1999b; Shirao et al., 2002). Caspase-3 cleaves ROCK-1 at the cleavage site DETD1113 whereas granzyme B cleaves the ROCK-2 at the C-terminus at IGLD1131, thus removing an inhibitory region (Fig. 4a) (Sebbagh et al., 2001, 2005). The activity of ROCKs is also negatively regulated by other small G-binding proteins such as RhoE, Gem, and Rad (Ward et al., 2002; Riento et al., 2003). RhoE binds to the N-terminal region of kinase domain of ROCK-1 (amino acids 1–420) and therefore interferes with the kinase activity and prevents GTPase-RhoA binding to Rho-binding domain (Fig. 4b) (Riento et al., 2003). Overexpression of Gem and Rad might inhibit respectively the downstream responses of ROCK-1 and ROCK-2, but the mechanism is not clearly demonstrated (Ward et al., 2002). In addition, the negative regulation of ROCK-mediated target effects by these small G-binding proteins are localized at the different cellular structure (RhoE in the Golgi and Gem and Rad in the cytoskeleton) (Bilan et al., 1998; Piddini et al., 2001). 4. Vascular downstream effect of Rho-kinase

Vascular Ca2+-independent contractivity is related to Ca2+ sensitization of VSMC that is dependent on an increase of MLC20 phosphorylation and a force of contractile myofilaments without eliciting a change in [Ca2+]i. Many cellular signaling pathways contribute to culminate the inhibition of myosin light chain phosphatase (MLCP) activity, increasing therefore the level of phosphorylated MLC20 (Somlyo & Somlyo, 2003). ROCKs, downstream effectors of GTPase-RhoA, phosphorylate the MLCP targeting subunit (MYPT1) at threonine (Thr)-696, Thr-853, and Thr-855 (Somlyo & Somlyo, 2003; Wilson et al., 2005; Knock et al., 2009), resulting in inhibition of MLCP activity and increasing of MLC20 phosphorylation (Feng et al., 1999a; Velasco et al., 2002). This in turn, enhances the actin binding and actin-induced ATPase activity of myosin, facilitating interaction of myosin with F-actin, then VSMC contractility (Fig. 5). However, the inhibition of MLCP activity might be mediated by a direct interaction of the small 17 kDa protein kinase C-potentiated myosin phosphatase inhibitor protein (CPI-17) with protein phosphatase 1 catalytic subunit (PP1c) of MLCP. Phosphorylation of CPI-17 by ROCKs at Thr-38 (Koyama et al., 2000), and protein kinase C (PKC) (Eto et al., 1997) also enhances the potency of CPI-17 for inhibiting MCLP activity (Takizawa et al., 2002), and VSMC contractility.

4.1. Rho-kinase and vascular smooth muscle cells (VSMC) 4.1.1. Rho-kinase and vascular smooth muscle cells contractility Vascular smooth muscle cell (VSMC) contractility is dependent on the level of phosphorylation of the 20 kDa myosin light chain (MLC20) that is determined by both Ca 2+-dependent and Ca 2+-independent mechanisms (Fig. 5). VSMC Ca 2+-dependent contractility is related to the concentration of Ca2+ in cytosolic ([Ca2+]i) which is regulated by the release of Ca2+ from the sarcoplasmic reticulum and the entry of Ca2+ from the extracellular space via voltage-dependent Ca2+ channels (VDCCs) or through non-selective cation channels (NSCCs) (VanBavel et al., 2002; Moosmang et al., 2003). Increase of [Ca2+]i in cytosolic promotes therefore the binding of Ca 2+ to a specific pool of calmodulin (CaM) tethered to myosin light chain kinase (MLCK) (Wilson et al., 2002). It results then to the activation of MLCK and MLCK-mediated phosphorylation of MLC20, thereby activating cross-bridge cycling and contraction.

4.1.2. Rho-kinase and vascular smooth muscle cells proliferation The proliferation of VSMCs is related to an activation of multiple pro-mitogenic signals, which interferes in the regulation of cell cycle (Assoian & Marcantonio, 1996). During the cell cycle, the progression through the G1 phase is regulated by phosphorylation and inactivation of retinoblastoma (Rb) proteins, which is promoted by the Cyclins (Cyclin A, D, and E), inducing an activation of cyclin-dependent kinases (cdk2, cdk4, and cdk6). These cdk-cyclin complexes are however, negatively regulated by the Cip/Kip family (p21Cip1, p27 Kip1, and p57Kip2) of Cyclin-dependent kinase inhibitors (CDKIs) (Morgan, 1995; Sherr & Roberts, 1995). Hence, the cyclin-dependent kinase inhibitor p27 Kip1 plays a crucial role in VSMC proliferation. There is increasing evidence that ROCK effectors downregulate p27 Kip1 expression, leading to the acceleration of cell cycle progression and VSMC proliferation (Laufs et al., 1999b; Sawada et al., 2000). The effect of ROCK inhibitors as antiproliferators has been also ascribed to

Fig. 3. The autoinhibition form of ROCKs. The Pleckstrin-homology (PH) and Rho-binding domain (RBD) bind to the N-terminus of the enzyme, forming an autoinhibitory loop.

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Fig. 4. a. The mechanism of positive regulation of ROCKs activity. Binding of GTPase-RhoA to Rho-binding domain (RBD) relieves the autoinhibitory blockage of kinase activity. Arachidonic acid (AA) and sphingosine phosphorylcholine (SPC) interact with the negative regulatory region at PH domain, disrupting the inhibitory property of ROCKs. Caspase-3 and granzyme B cleave ROCKs at cleavage sites, removing the inhibitory regions. b. The mechanism of negative regulation of ROCKs activity. RhoE binds to the N-terminal region of kinase domain of ROCKs (ROCK-1) prevents GTPase-RhoA binding to Rho-binding domain (RBD).

the upregulation of p27Kip1 expression (Sawada et al., 2000; Kanda et al., 2005). In addition, the role of ROCKs in the proliferation of VSMC mediated by platelet-derived growth factor (PDGF) has been demonstrated by a previous study (Kamiyama et al., 2003). Inhibition of ROCKs abolishes the PDGF-induced activation of extracellular-regulated kinase 1/2 (ERK1/2) and proliferation of VSMCs. Moreover, inhibition of ROCKs also suppresses VSMC proliferation mediated by G-protein-coupled receptor-stimulated cell proliferation which is promoted by many mediators such as thrombin, urotensin-II, and angiotensin-II (Seasholtz et al., 1999; Yamakawa et al., 2000; Sauzeau et al., 2001). 4.2. Rho-kinase and endothelial cells (EC) 4.2.1. Rho-kinase and endothelial nitric oxide synthase/nitric oxide signaling In endothelium, nitric oxide (NO) is synthesized from the conversion of L-arginine to L-citrulline in the presence of endothelial nitric oxide synthase (eNOS) by using oxygen, NADPH, and substrates (Moncada et al., 1989). After diffusing into the VSMCs, NO activates soluble guanylate cyclase (sGC), which catalyzes the formation of

guanosine monophosphate (cGMP) and the subsequent activation of cGMP-dependent protein kinase (cGK). The effect of NO/cGK cascade signalization in VSMC relaxation results from the decrease of [Ca 2+]i intracellular due to the activation of the sarcoplasmatic Ca2+-ATPase, reducing Ca2+ release from intracellular store and the phosphorylation and activation of MLP20. In addition, in VSMCs, the role of eNOS/NO signalization as antiproliferation has been demonstrated (Emerson et al., 1999; Keil et al., 2002). Recent studies showed that Rho-kinase/ROCKs might negatively regulate eNOS expression, eNOS activity, and NO bioavailability (Laufs & Liao, 1998; Laufs et al., 1999a; Takemoto et al., 2002; Rikitake & Liao, 2005). Inhibition of ROCKs increases eNOS mRNA stability and eNOS expression (Rikitake et al., 2005). ROCK inhibitors also enhance phosphorylation and activation of the Akt/phosphatidylinositol-3 kinase (PI-3K) pathway, leading to increase NO production (Wolfrum et al., 2004). In addition, the pleiotropic effect of statins, a nonselective ROCKs inhibitors, on upregulation of eNOS has been demonstrated (Ni et al., 2001; Girgis et al., 2003; Nishimura et al., 2003). Statins upregulate eNOS/NO signalization

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Fig. 5. Ca2+ sensitization of VSMC mediated by RhoA/Rho-kinase signaling pathway. In basal state of VSMCs, there is a balance between vasoconstriction and vasodilatation that is controlled by the activity of MLCK and MLCP. When Rho-kinase agonists are coupled on GPCR, it converts GDP-RhoA (inactive form) to GTP-RhoA (active form). GTP-RhoA translocates into the membrane and activates its downstream effectors: Rho-kinase or ROCKs. ROCKs increase the vasoconstriction by inhibiting the activity of MLCP via the phosphorylation of this enzyme.

via the inhibition of geranylgeranylation of the small G-protein Rho, and translocate inactive Rho form the cytosol to the membrane (Liao & Laufs, 2005). 4.2.2. Endothelial nitric oxide synthase/nitric oxide signaling and GTPaseRhoA/Rho-kinase On the other hand, the effect of eNOS/NO signalization on RhoA/ Rho-kinase pathway has been recently studied. Previous studies founded that NO through cGMP/cGK might serine-phosphorylate RhoA leading to decrease the association of RhoA protein on cell membrane (Sauzeau et al., 2000; Begum et al., 2002; Chitaley & Webb, 2002; Krepinsky et al., 2003). As a result, it reduced RhoA-GTP active form, and therefore, decreased the activity of downstream target effects of ROCKs. Sauzeau et al. (Sauzeau et al., 2000) showed that exogenous NO attenuated Ca 2+-dependent ROCKs sensitization of blood vessel contraction by inhibiting RhoA translocation from the cytosol to membrane in VSMCs via cGK pathway. Moreover, NO might also inhibit RhoA/ROCKs independently of cGMP by S-nitrosating RhoA via interaction with cysteine to form nitrothiols (Hess et al., 2001; Stamler et al., 2001; Zuckerbraun et al., 2007). 5. Role of Rho-kinase signaling in pulmonary hypertension 5.1. In animal models The role of RhoA/Rho-kinase pathway in PAH has been principally studied in chronic hypoxia (CH)-induced PH and monocrotaline (MCT)-induced PH (Table 1). Chronic hypoxia due to different disorders or pathologies, is one of the major causes of PH. Exposure to chronic hypoxia induces structural and functional changes in pulmonary arterial bed (Rabinovitch et al., 1979; Pierson, 2000) leading to endothelial dysfunction (Higenbottam & Laude, 1998), and modification of pulmonary vascular contractile properties (Yuan et al., 1998) as described above. Furthermore, in hypoxia-induced PH, the balance of vasoconstriction–vasodilatation tonus has been impaired by other

vasoconstrictors such as ET-1, 5-HT, Ang II. These vasoconstrictors also mediate RhoA/Rho-kinase pathway (Barman, 2007; Homma et al., 2007), and it in turn, is involved in downregulation of eNOS signalization, VSMC hyperconstriction (Batchelor et al., 2001; Sakurada et al., 2001; Nagaoka et al., 2004, 2006; McNamara et al., 2008; Desbuards et al., 2009), and pulmonary vascular remodeling (Laufs et al., 1999a; Sauzeau et al., 2001; Keil et al., 2002). It is known that distal muscularization of pulmonary circulation has been increased during chronic hypoxia and RhoA/Rho-kinase pathway is involved in growth and hypertrophy of SMC via the mitogenic effectors (ET-1, 5-HT) in pulmonary vascular remodeling (Emerson et al., 1999; Fagan et al., 1999; Aznar and Lacal, 2001; Keil et al., 2002). In animal model, PH can be achieved by injection with a single subcutaneous or intraperitoneal of monocrotaline (MCT). Although the exact mechanism though which MCT-induced PH is not well understood. It is suggested that injection of MCT-induced severe or lethal PH is mediated by its direct toxic effect on endothelial cells and dramatic accumulation of mononuclear inflammatory cells, particularly macrophage, in the small intraacinar vessels (Wilson et al., 1989; Jasmin et al., 2001). The role of Rho-kinase in MCT-induced PH in rats has been firstly demonstrated by Khan et al. (2005). In this study, the authors showed that while pulmonary arterial contraction induced by noradrenaline was attenuated and due to poor-coupling to G-protein-coupled receptor, the Rho-kinase activity was increased with principally ROCK-2. Increase of Rho-kinase activity, mediated by HMG-CoA reductase and cleaved caspase-3, is also involved in MCT-induced PH in pneumonectomized rats (Homma et al., 2008). Recent study showed that increases asymmetric dimethylarginine (ADMA) which is involved in vascular remodeling in MCT-induced PH, is mediated by Rho-kinase activity (Li et al., 2010). Rho-kinase signaling pathway also plays a important role in the pathogenesis of other models of PH. Li F et al. demonstrated that Wister rats with carotid-external jugular vein communication, a model of high-pressure systemic–to-pulmonary shunts, developed PH after 4 wk with excessive vascular remodeling at week 8 (Li et al., 2007). In

S. Duong-Quy et al. / Pharmacology & Therapeutics 137 (2013) 352–364 Table 1 The role of Rho-kinase signaling pathway in pulmonary hypertension. Authors, Year

PH model

Involved role of Rho-kinase signaling in PH

(Li et al., 2010) (Do et al., 2009)

MCT-induced PH in rats

(Guilluy et al., 2009)

iPAH patients

PASMCs proliferation mediated by ADMA Impaired endothelium-dependent relaxation; serotonin-induced hypercontraction 5-HT -mediated PASMCs proliferation and platelet activation PH due to discontinuation of anorexia

iPAH patients

Hypoxic associated with discontinuated dexfenfluramine-induced PH in rats (Gien et al., Partial ligation of the ductus 2008) arteriosus in utero (PPHN) sheep (Homma et MCT-induced PH in al., 2008) pneumonectomized rats (McNamara CH- and bleomycin-induced PH in rats et al., 2008) (Hemnes et Bleomycin-induced PH in rats al., 2008) (Homma et CH-induced PH in rats al., 2007) (Barman, CH-induced PH in rats 2007) (Oka et al., Severe occlusive PAH in rats 2007) (Li et al., High flow-induced PH in rats 2007) PH of Denver FHR (Nagaoka et al., 2006) (Khan et al., MCT-induced PH in rats 2005) CH-induced PH in rats (Nagaoka et al., 2004) (Desbuards et al., 2009)

Impaired angiogenesis; downregulated eNOS and NO PH mediated by HMG-CoA reductase and cleaved caspase-3 Vasoconstriction

ROS-mediated lung fibrosis and NOS uncoupling Vasoconstriction in response to KCl mediated by ET-1 and 5-HT ET-1-induced vasoconstriction Angioproliferation in pulmonary arterioles; vasoconstriction Vascular remodeling Sustained vasoconstriction

NA-induced PA contraction Vasoconstriction

MCT, monocrotaline; PH, pulmonary hypertension; ADMA, asymmetric dimethylarginine; PASMCs, pulmonary arterial smooth muscle cells; iPAH, idiopathic pulmonary arterial hypertension; ROS, reactive oxygen species; 5-HT, serotonin; PPHN, persistent pulmonary hypertension of the newborn; CH, chronic hypoxia; ET-1, endothelin-1; FHR, fawn hooded rat; NA, noradrenalin.

this study, Rho-kinase signaling pathway was involved in increased proliferation and decreased apoptosis of SMCs. In severe PH model induced by hypoxia associated with injected Sugen 5416, a VEGF receptor inhibitor, Oka et al. (2007) founded that Rho-kinase activity had a crucial role in the development of occlusive pulmonary vascular lesions and sustained vasoconstriction. The role of Rho-kinase signaling pathway in bleomycin-induced PH has been demonstrated (Hemnes et al., 2008). It is speculated that in this model, Rho-kinase activity is mediated by enhanced reactive oxygen species (ROS), resulting lung fibrosis and NOS uncoupling. Another role of Rho-kinase in animal model with PH has been also reported (Gien et al., 2008). 5.2. In humans Although the role of Rho-kinase signaling pathway in pathogenesis of PH has been confirmed in diverse animal models, its role in human pulmonary hypertension is not well studied. The first published study in this field realized by Guilluy et al. (2009), demonstrated that RhoA/Rho-kinase activity was increased in patients with idiopathic pulmonary arterial hypertension (iPAH). The upregulation of RhoA/Rho-kinase in these patients has been mediated by 5-HT via the serotonylation of Rho protein, contributing to the proliferation of pulmonary arterial SMCs. In the study from patients with pulmonary hypertension who underwent lung transplantation, Do et al. (2009)

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showed that Rho-kinase activity was correlated to severity (mean pulmonary arterial pressure, mPAP) and evolution of the disease. In this study, Rho-kinase activity was involved in pulmonary arterial hypercontraction mediated by PGK and endothelial dysfunction. 6. Rho-kinase inhibitors in the treatment of pulmonary hypertension 6.1. In animal models 6.1.1. Y-27632 in the treatment of pulmonary hypertension Y-27632 [−R-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride]] is one of the compounds derived from pyridine with potent ROCK inhibitory effect (Uehata et al., 1997). Y-27632 is a non-specific inhibitor of ROCKs, competing with ATP for binding to ROCK catalytic sites (Ishizaki et al., 2000). It has also a potent inhibitory effect of Rho-dependent protein kinase C (Uehata et al., 1997). In the initial study on the effect of inhibition of Rho-kinase in mice with CH-induced PAH, Fagan et al. (2004) reported that Y-27632 decreased pulmonary vasoconstriction via mediating Ca 2+ sensitization and attenuated the risk of developing PH and vascular remodeling. The study showed that Y-27632 at 30 mg −1.day −1 reduced right ventricle systolic pressure (RVSP), right ventricle hypertrophy (RV hypertrophy), and neomuscularization of the distal pulmonary vasculature. Particularly, treatment with Y-27632 increased the expression of eNOS protein in lung tissues of CH-induced PH. Many other studies also showed that in CH-induced PH in rats, Y-27632 attenuated significantly hemodynamic changes and vascular remodeling in treated rats in comparison with non treated group (McNamara et al., 2008; Xu et al., 2010; Ziino et al., 2010). Recently, Vanderpool et al. (2011) showed that, by using isolated lung of CH-induced PH, Y-27632 decreased the pulmonary arterial impedance, the diameter of main pulmonary arterial, and the right pulmonary compliance. However, Y-27632 is not a high selective pulmonary vasodilator because of its effects on systemic circulation when used intravenously or orally. To avoid the systemic vasodilator effect of Y-27632, Nagaoka et al. (2005) demonstrated that inhalation of aerosolized Y-27632 had a selective effect by reducing mean pulmonary arterial pressure (mPAP) without systemic effect in chronic hypoxia Sprague–Dawley Rat (SDR). The selective pulmonary hypotension effect of aerosolized Y-27632 lasted more than 5 h after 5 min of inhalation. Especially, its effect was stronger than that with inhalation of NO. 6.1.2. Fasudil in the treatment of pulmonary hypertension Fasudil (HA-1077) is the first approved ROCK for clinical use in the treatment of ischemia-induced brain damage (Toshima et al., 2000; Satoh et al., 2001; Rikitake et al., 2005). Fasudil is a selective ROCK inhibitor, competing with ATP for the binding to the kinase (Davies et al., 2000). After oral administration, fasudil is metabolized in hydroxyl fasudil (HA-1100), having more selective inhibitory effect on ROCKs (Shimokawa et al., 1999). Fasudil is more effective than Y-27632 in prevention of CH-induced PH (Shimokawa, 2002; Shimokawa et al., 2002; Abe et al., 2006). The first use of fasudil in the treatment of PH in animal model has been demonstrated by Abe et al. (2004). In this study, the long-term treatment with fasudil improved hemodynamic parameters (PAP, RVSP, RV hypertrophy), pulmonary vascular remodeling with suppression of VSMC proliferation and enhancing of VSMC apoptosis in MCT-induced PH in rats. Fasudil ameliorated endothelial dysfunction and VSMC hypercontraction. The beneficial effect of fasudil in hemodynamic parameters in MCTinduced PH has been demonstrated by other studies (Jiang et al., 2007; Mouchaers et al., 2010). The role of fasudil in the treatment of PH also has been demonstrated in other model of PH (Table 2). In Wistar rats with high flow-induced PH (Li et al., 2007), treatment with fasudil suppressed Rho-kinase

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hyperactivity, leading to improve PAP, RV hypertrophy, and pulmonary vascular remodeling. These results were similar to CH-induced PH or severe occlusive PH, and left-ventricular dysfunction-induced PH (Yamakawa et al., 2000; Guilluy et al., 2005; Abe et al., 2006; Nagaoka et al., 2006; Oka et al., 2007; Ziino et al., 2010; Dai et al., 2011). Particularly, fasudil improved the expression of eNOS (Abe et al., 2006) and normalized pulmonary vascular resistance (PVR) in PH refractory to

Table 2 Effects of Rho-kinase inhibitors in PH animal model. Authors, Year

PH model

(Vanderpool et al., 2011) (Dai et al., 2011)

Y-27632 (10− 5M , isolated mouse lungs) Fasudil Left-ventricular dysfunction-induced (30 mg/kg) PH in rats CH-induced PH in Y-27632 juvenile rats (15 mg/kg)

(Xu et al., 2010)

Rho-kinase inhibitors (daily dose)

CH-induced PH in mice

(Dahal et al., MCT-induced PH in 2010) rats; CH-induced PH in mice (Mouchaers MCT-induced PH in rats et al., 2010) (Ziino et al., CH-induced PH in 2010) neonatal rats

Azaindole-1 (10–30 mg/kg) Fasudil (40 mg/kg)

(McNamara et al., 2008)

CH- and bleomycin-induced PH in rats

(Tawara et al., 2007)

MCT-induced PH in rats

(Oka et al., 2007)

Severe occlusive PAH in rats

(Jiang et al., 2007) (Girgis et al., 2007)

MCT-induced PH in rats CH-induced PH in rats

(Li et al., 2007)

High flow-induced PH in rats

Y-27632 (10 mg/kg) Fasudil (20 mg/kg) Y-27632 (15 mg/kg) Fasudil (30 mg/kg) Fasudil (30 mg/ kg) associated with Beraprost sodium Fasudil (10 mg/ kg, intravenous administration) Fasudil (10–30 mg/kg) Simvastatine (non selective inhibitor) Fasudil (30 mg/kg)

(Abe et al., 2006)

CH-induced PH in mice

Fasudil (100 mg/kg)

(Nagaoka et al., 2006)

PH of Denver FHR

Fasudil (30 mg/kg)

(Guilluy et al., 2005) (Nagaoka et al., 2005)

CH-induced PH in rats CH-induced PH in rats MCT-induced PH in rats

(Fagan et al., 2004) (Abe et al., 2004)

CH-induced PH in mice MCT-induced PH in rats

Fasudil (30 mg/kg) Y-27632 (30 mg/kg) Inhaled Y-27632 and fasudil Y-27632 (30 mg/kg) Fasudil (30– 100 mg/kg)

Effect of Rho-kinase inhibitors Decreased: PA impedance, diameter of main PA, and right PA compliance Improved: mPAP, RV hypertrophy, PA medial thickness Attenuated: RV hypertrophy, PA wall remodeling; normalized: RVSF Improved: RVSP, TPR, RV hypertrophy; anti-PASMC proliferation Reduced: mPAP, PVR, RVSP, RV hypertrophy Attenuated: hemodynamic and structural changes

Normalized: PVR (PH refractory to NO)

Decreased: mPAP, RV hypertrophy, medial thickening (synergistic effect) Decreased: RVSP, vasoconstriction Increased: CO Reduced: mPAP

nitric oxide (NO) (McNamara et al., 2008). In Fawn-Hooded Rat (FHR) with severe PH when raised for the first weeks of life in the mild hypoxia of Denver's altitude (Nagaoka et al., 2006), long-term treatment with fasudil reduced the development of PH and improved lung dysplasia in rat pups by decreasing alveolar size and increasing pulmonary vascular density. Moreover, fasudil might be used in combination with other vasodilators in the treatment of PH. Tawara et al. (2007) showed that in MCT-induced PH in rats, when compared with monotherapy, the combination of fasudil and prostacyclin analogue (beraprost sodium) significantly improved mPAP, RV hypertrophy, and pulmonary arterial medial thickness without any adverse effects. This result suggested that combined therapy between a Rho-kinase inhibitor with other vasodilators might exert more beneficial effects in PH treatment due to the additive and synergic effects of combined treatment. In addition, fasudil when used by inhalation is as effective as inhaled Y-27632 in the treatment of PH. In CH- and MCT-induced PH in rats, inhaled fasudil reduced significantly mPAP, without any significant change in systemic arterial pressure and cardiac index or heart rate (Nagaoka et al., 2005). This route of administration is one of the advantages of fasudil in the treatment of PH and it should be explored in the future.

6.1.3. Other Rho-kinase inhibitors in the treatment of pulmonary hypertension Although many other new Rho-kinase inhibitors have been developed, their role in the treatment of PH has not been completely studied. Recently, the therapeutic efficacy of azaindole-1, a potent ROCK inhibitor with highly specific ATP-competition, in MCT- and CH-induced PH in rodent has been demonstrated (Dahal et al., 2010). Oral administration of azaindole-1 improved the hemodynamic, RV hypertrophy and pulmonary vascular remodeling. In this study, azaindole-1 had a potent effect improving a hypoxic pulmonary vasoconstriction and a pulmonary arterial SMC proliferation. Beside the effects of selective Rho-kinase inhibitors in the treatment of PH, the role of statin, a non selective Rho-kinase inhibitor with pleiotropic effect, in the treatment of PH has been demonstrated (Girgis et al., 2007).

6.2. In humans Reduced: mPAP, RV hypertrophie, medial thickening Suppressed: PASMC proliferation Attenuated: RV hypertrophy Improved: mPAP, RV hypertrophie, eNOS expression Reduced: elevated PAP Improved: alveolarization vascularization Reduced: PAP, RV hypertrophy Decreased: mPAP

Decreased: RVSP, RV hypertrophie Improved: survival, RVSP, RV hypertrophie, medial thickening, endothelial dysfunction

PH, pulmonary hypertension; CH, chronic hypoxia; PA, pulmonary artery; SMCs, smooth muscle cells; MCT, monocrotaline; RV, right ventricle; RVSF, right ventricular systolic function; RVSP, right ventricular systemic pressure; mPAP, mean pulmonary arterial pressure; TPR, total pulmonary resistance; PVR, pulmonary vascular resistance.

Successful results of Rho-kinase inhibitors in the treatment of PH in animal model encourage many researchers try to study it in human. The first clinical essays of Rho-kinase inhibitors in treatment of PH have been initiated by Fukumoto et al. (2005) in a small number of enrolled patients (9 patients, mean age of 53 years). The results of this study showed that intravenous administration of fasudil (30 mg for 30 minutes) decreased PAP and increased cardiac index (CI). It also reduced PVR significantly without any side effect on systemic pressure. This result is similar to what reported by Ishikura et al. in patients with idiopathic pulmonary arterial hypertension (iPAH) and associated PAH (Ishikura et al., 2006). Recently, the acute beneficial effects of fasudil on hemodynamic in the treatment of congenital heart disease with left-to-right shunt-induced PH in young patients have been demonstrated by a prospective study (12 patients, mean age of 12.3 years) (Li et al., 2009). In this study, intravenous injection of fasudil (30 mg over 30 min) significantly decreased PAP and PVR and increased cardiac output, whereas systemic arterial pressure were slightly reduced as compared to baseline value. Fujita et al. showed that the use of fasudil by inhalation in the treatment of PH (iPAH, aPAH, and PAH due to left heart dysfunction) decreased mPAP and the ratio of pulmonary vascular resistance/systemic vascular resistance (Fujita et al., 2010). However, the long term effect of fasudil in patients with PH as monotherapy or in combination with other treatment has not been demonstrated (Table 3).

S. Duong-Quy et al. / Pharmacology & Therapeutics 137 (2013) 352–364 Table 3 Effects of Rho-kinase inhibitors in patients with PAH. Authors, Year

PH model

Rho-kinase inhibitors

Effect of Rho-kinase inhibitors

(Fujita et al., 2010)

Patients with PAH (iPAH, aPAH, PAH due to LHD) Left-to-right shunt-induced PH

Fasudil (inhalation: 30 mg/ 10 min)

Decreased: mPAP, PVR/SVR

Fasudil (intravenous administration: 30 mg/ 30 min)

Decreased: PASP, PVR, PVR/ SVR Increased: CI Decreased: mPAP, TPR Increased: CI Decreased: mPAP, PVR Increased: CI

(Li et al., 2009)

(Ishikura et Patients with PAH al., 2006) ( iPAH, aPAH) (Fukumoto et al., 2005)

Patients with severe PAH

Fasudil (intravenous administration: 30 mg/ 30 min) Fasudil (intravenous administration: 30 mg/ 30 min)

PAH, pulmonary arterial hypertension; iPAH, idiopathic PAH; aPAH, associated PAH; LHD, left heart disease; mPAP, mean pulmonary arterial pressure; TPR, total pulmonary resistance; PVR, pulmonary vascular resistance; SVR, systemic vascular resistance; PASP, pulmonary arterial systolic pressure; CI, cardiac index.

7. Conclusion More than thirty years after the first meeting of PH in Geneva, up to now, PH is still an incurable disease with dramatic survival. Despite the combination of available vasodilator drugs at early stage of disease, the prognostic of PH remains unfortunately the same as some progressed cancer. The new findings on pathophysiology and pathobiology of PH help physicians improving advanced knowledge on pathogenesis of this disease. Recent discovery on the role of RhoA/Rho-kinase signaling pathway in patients with PH and the effects of Rho-kinase inhibitors in vivo promise a novel pharmacological treatment of PH. These selective Rho-kinase inhibitors are aimed to improve the pulmonary arterial relaxation and remodeling. However, the long-term outcome of clinical trials of Rho-kinase inhibitors in patients with PH will be necessary in the future to confirm its effects. Conflict of interest statement The undersigned authors, Sy Duong‐Quy, Yihua Bei, Zhongmin Liu, and Anh Tuan Dinh‐Xuan declare that there are no conflict of interest related to this work. References Abe, K., Shimokawa, H., Morikawa, K., Uwatoku, T., Oi, K., Matsumoto, et al. (2004). Long-term treatment with a Rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hypertension in rats. Circ Res 94, 385–393. Abe, K., Tawara, S., Oi, K., Hizume, T., Uwatoku, T., Fukumoto, Y., et al. (2006). Long-term inhibition of Rho-kinase ameliorates hypoxia-induced pulmonary hypertension in mice. J Cardiovasc Pharmacol 48, 280–285. Alberts, A. S., Bouquin, N., Johnston, L. H., & Treisman, R. (1998). Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein beta subunits and the yeast response regulator protein Skn7. J Biol Chem 273, 8616–8622. Amano, M., Chihara, K., Nakamura, N., Kaneko, T., Matsuura, Y., & Kaibuchi, K. (1999). The COOH terminus of Rho-kinase negatively regulates rho-kinase activity. J Biol Chem 274, 32418–32424. Amano, M., Fukata, Y., & Kaibuchi, K. (2000). Regulation and functions of Rho-associated kinase. Exp Cell Res 261, 44–51. Assoian, R. K., & Marcantonio, E. E. (1996). The extracellular matrix as a cell cycle control element in atherosclerosis and restenosis. J Clin Invest 98, 2436–2439. Aznar, S., & Lacal, J. C. (2001). Rho signals to cell growth and apoptosis. Cancer Lett 165, 1–10. Balabanian, K., Foussat, A., Dorfmüller, P., Durand-Gasselin, I., Capel, F., Bouchet-Delbos, L., et al. (2002). CX(3)C chemokine fractalkine in pulmonary arterial hypertension. Am J Respir Crit Care Med 165, 1419–1425. Barman, S. A. (2007). Vasoconstrictor effect of endothelin-1 on hypertensive pulmonary arterial smooth muscle involves Rho-kinase and protein kinase C. Am J Physiol Lung Cell Mol Physiol 293, L472–L479.

361

Batchelor, T. J., Sadaba, J. R., Ishola, A., Pacaud, P., Munsch, C. M., & Beech, D. J. (2001). Rho-kinase inhibitors prevent agonist-induced vasospasm in human internal mammary artery. Br J Pharmacol 132, 302–308. Begum, N., Sandu, O. A., & Duddy, N. (2002). Negative regulation of rho signaling by insulin and its impact on actin cytoskeleton organization in vascular smooth muscle cells: Role of nitric oxide and cyclic guanosine monophosphate signaling pathways. Diabetes 51, 2256–2263. Bilan, P. J., Moyers, J. S., & Kahn, C. R. (1998). The ras-related protein rad associates with the cytoskeleton in a non-lipid-dependent manner. Exp Cell Res 242, 391–400. Channick, R. N., Simonneau, G., Sitbon, O., Robbins, I. M., Frost, A., Tapson, V. F., et al. (2001). Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: A randomised placebo-controlled study. Lancet 358, 1119–1123. Chevrier, V., Piel, M., Collomb, N., Saoudi, Y., Frank, R., Paintrand, M., et al. (2002). The Rho-associated protein kinase p160ROCK is required for centrosome positioning. J Cell Biol 157, 807–817. Chitaley, K., & Webb, R. C. (2002). Nitric oxide induces dilation of rat aorta via inhibition of rho-kinase signaling. Hypertension 39, 438–442. Christman, B. W., McPherson, C. D., Newman, J. H., King, G. A., Bernard, G. R., Groves, B. M., et al. (1992). An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension. N Engl J Med 327, 70–75. D'Alonzo, G. E., Barst, R. J., Ayres, S. M., Bergofsky, E. H., Brundage, B. H., Detre, K. M., et al. (1991). Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Ann Intern Med 115, 343–349. Dahal, B. K., Kosanovic, D., Pamarthi, P. K., Sydykov, A., Lai, Y. -J., Kast, R., et al. (2010). Therapeutic efficacy of azaindole-1 in experimental pulmonary hypertension. Eur Respir J 36, 808–818. Dai, Z. -K., Wu, B. -N., Chen, I. -C., Chai, C. -Y., Wu, J. -R., Chou, S. -H., et al. (2011). Attenuation of pulmonary hypertension secondary to left ventricular dysfunction in the rat by Rho-kinase inhibitor fasudil. Pediatr Pulmonol 46, 45–59. Davies, S. P., Reddy, H., Caivano, M., & Cohen, P. (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351, 95–105. Desbuards, N., Antier, D., Rochefort, G. Y., Apfeldorfer, C. S., Schenck, E., Hanton, G., et al. (2009). Dexfenfluramine discontinuous treatment does not worsen hypoxia-induced pulmonary vascular remodeling but activates RhoA/ROCK pathway: Consequences on pulmonary hypertension. Eur J Pharmacol 602, 355–363. Dingemans, K. P., & Wagenvoort, C. A. (1978). Pulmonary arteries and veins in experimental hypoxia. An ultrastructural study. Am J Pathol 93, 353–368. Do, e Z., Fukumoto, Y., Takaki, A., Tawara, S., Ohashi, J., Nakano, M., et al. (2009). Evidence for Rho-kinase activation in patients with pulmonary arterial hypertension. Circ J 73, 1731–1739. Doran, J. D., Liu, X., Taslimi, P., Saadat, A., & Fox, T. (2004). New insights into the structure-function relationships of Rho-associated kinase: A thermodynamic and hydrodynamic study of the dimer-to-monomer transition and its kinetic implications. Biochem J 384, 255–262. Dorfmüller, P., Perros, F., Balabanian, K., & Humbert, M. (2003). Inflammation in pulmonary arterial hypertension. Eur Respir J 22, 358–363. Eddahibi, S., Chaouat, A., Morrell, N., Fadel, E., Fuhrman, C., Bugnet, A. -S., et al. (2003). Polymorphism of the serotonin transporter gene and pulmonary hypertension in chronic obstructive pulmonary disease. Circulation 108, 1839–1844. Eddahibi, S., Humbert, M., Fadel, E., Raffestin, B., Darmon, M., Capron, F., et al. (2001). Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension. J Clin Invest 108, 1141–1150. Emerson, M., Momi, S., Paul, W., Alberti, P. F., Page, C., & Gresele, P. (1999). Endogenous nitric oxide acts as a natural antithrombotic agent in vivo by inhibiting platelet aggregation in the pulmonary vasculature. Thromb Haemost 81, 961–966. Eto, M., Senba, S., Morita, F., & Yazawa, M. (1997). Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: Its specific localization in smooth muscle. FEBS Lett 410, 356–360. Fagan, K. A., Fouty, B. W., Tyler, R. C., Morris, K. G., Jr., Hepler, L. K., Sato, K., et al. (1999). The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresponsive to mild hypoxia. J Clin Invest 103, 291–299. Fagan, K. A., Oka, M., Bauer, N. R., Gebb, S. A., Ivy, D. D., Morris, K. G., et al. (2004). Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase. Am J Physiol Lung Cell Mol Physiol 287, L656–L664. Feng, J., Ito, M., Ichikawa, K., Isaka, N., Nishikawa, M., Hartshorne, D. J., et al. (1999a). Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J Biol Chem 274, 37385–37390. Feng, J., Ito, M., Kureishi, Y., Ichikawa, K., Amano, M., Isaka, N., et al. (1999b). Rho-associated kinase of chicken gizzard smooth muscle. J Biol Chem 274, 3744–3752. Foletta, V. C., Lim, M. A., Soosairajah, J., Kelly, A. P., Stanley, E. G., Shannon, M., et al. (2003). Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. J Cell Biol 162, 1089–1098. Fujisawa, K., Madaule, P., Ishizaki, T., Watanabe, G., Bito, H., Saito, Y., et al. (1998). Different regions of Rho determine Rho-selective binding of different classes of Rho target molecules. J Biol Chem 273, 18943–18949. Fujita, H., Fukumoto, Y., Saji, K., Sugimura, K., Demachi, J., Nawata, J., et al. (2010). Acute vasodilator effects of inhaled fasudil, a specific Rho-kinase inhibitor, in patients with pulmonary arterial hypertension. Heart Vessels 25, 144–149. Fukata, Y., Amano, M., & Kaibuchi, K. (2001). Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci 22, 32–39. Fukumoto, Y., Matoba, T., Ito, A., Tanaka, H., Kishi, T., Hayashidani, S., et al. (2005). Acute vasodilator effects of a Rho-kinase inhibitor, fasudil, in patients with severe pulmonary hypertension. Heart 91, 391–392.

362

S. Duong-Quy et al. / Pharmacology & Therapeutics 137 (2013) 352–364

Furuya, Y., Satoh, T., & Kuwana, M. (2010). Interleukin-6 as a potential therapeutic target for pulmonary arterial hypertension. Int J Rheumatol 2010, 720305. Galiè, N., Hoeper, M. M., Humbert, M., Torbicki, A., Vachiery, J. -L., Barbera, J. A., et al. (2009). Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Respir J 34, 1219–1263. Galié, N., Manes, A., & Branzi, A. (2004). The endothelin system in pulmonary arterial hypertension. Cardiovasc Res 61, 227–237. Giaid, A., & Saleh, D. (1995). Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 333, 214–221. Gien, J., Seedorf, G. J., Balasubramaniam, V., Tseng, N., Markham, N., & Abman, S. H. (2008). Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro. Am J Physiol Lung Cell Mol Physiol 295, L680–L687. Girgis, R. E., Li, D., Zhan, X., Garcia, J. G. N., Tuder, R. M., Hassoun, P. M., et al. (2003). Attenuation of chronic hypoxic pulmonary hypertension by simvastatin. Am J Physiol Heart Circ Physiol 285, H938–H945. Girgis, R. E., Mozammel, S., Champion, H. C., Li, D., Peng, X., Shimoda, L., et al. (2007). Regression of chronic hypoxic pulmonary hypertension by simvastatin. Am J Physiol Lung Cell Mol Physiol 292, L1105–L1110. Guilluy, C., Eddahibi, S., Agard, C., Guignabert, C., Izikki, M., Tu, L., et al. (2009). RhoA and Rho kinase activation in human pulmonary hypertension: role of 5-HT signaling. Am J Respir Crit Care Med 179, 1151–1158. Guilluy, C., Sauzeau, V., Rolli-Derkinderen, M., Guérin, P., Sagan, C., Pacaud, P., et al. (2005). Inhibition of RhoA/Rho kinase pathway is involved in the beneficial effect of sildenafil on pulmonary hypertension. Br J Pharmacol 146, 1010–1018. Hassoun, P. M., Mouthon, L., Barberà, J. A., Eddahibi, S., Flores, S. C., Grimminger, F., et al. (2009). Inflammation, growth factors, and pulmonary vascular remodeling. J Am Coll Cardiol 54(1 Suppl.), S10–S19. Hatano, S., & Strasser, T. (1975). Primary pulmonary hypertension. Report on a WHO Meeting. October 15–17, 1973 (pp. 1–45). Geneva: World Health Organization. Hemnes, A. R., Zaiman, A., & Champion, H. C. (2008). PDE5A inhibition attenuates bleomycin-induced pulmonary fibrosis and pulmonary hypertension through inhibition of ROS generation and RhoA/Rho kinase activation. Am J Physiol Lung Cell Mol Physiol 294, L24–L33. Hess, D. T., Matsumoto, A., Nudelman, R., & Stamler, J. S. (2001). S-nitrosylation: spectrum and specificity. Nat Cell Biol 3, E46–E49. Higenbottam, T. W., & Laude, E. A. (1998). Endothelial dysfunction providing the basis for the treatment of pulmonary hypertension: Giles F. Filley lecture. Chest 114(1 Suppl.), 72S–79S. Hoeper, M. M., & Dinh-Xuan, A. T. (2004). Combination therapy for pulmonary arterial hypertension: still more questions than answers. Eur Respir J 24, 339–340. Homma, N., Nagaoka, T., Karoor, V., Imamura, M., Taraseviciene-Stewart, L., Walker, L. A., et al. (2008). Involvement of RhoA/Rho kinase signaling in protection against monocrotaline-induced pulmonary hypertension in pneumonectomized rats by dehydroepiandrosterone. Am J Physiol Lung Cell Mol Physiol 295, L71–L78. Homma, N., Nagaoka, T., Morio, Y., Ota, H., Gebb, S. A., Karoor, V., et al. (2007). Endothelin-1 and serotonin are involved in activation of RhoA/Rho kinase signaling in the chronically hypoxic hypertensive rat pulmonary circulation. J Cardiovasc Pharmacol 50, 697–702. Humbert, M., Monti, G., Brenot, F., Sitbon, O., Portier, A., Grangeot-Keros, L., et al. (1995). Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am J Respir Crit Care Med 151, 1628–1631. Humbert, M., Morrell, N. W., Archer, S. L., Stenmark, K. R., MacLean, M. R., Lang, I. M., et al. (2004). Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol 43(12 Suppl. S), 13S–24S. Isern, R. A., Yaneva, M., Weiner, E., Parke, A., Rothfield, N., Dantzker, D., et al. (1992). Autoantibodies in patients with primary pulmonary hypertension: association with anti-Ku. Am J Med 93, 307–312. Ishikura, K., Yamada, N., Ito, M., Ota, S., Nakamura, M., Isaka, N., et al. (2006). Beneficial acute effects of rho-kinase inhibitor in patients with pulmonary arterial hypertension. Circ J 70, 174–178. Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatsu, A., Fujita, A., et al. (1996). The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J 15, 1885–1893. Ishizaki, T., Uehata, M., Tamechika, I., Keel, J., Nonomura, K., Maekawa, M., et al. (2000). Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol 57, 976–983. Ito, K., Ichiki, T., Ohi, K., Egashira, K., Ohta, M., Taguchi, K., et al. (2003). Pulmonary capillary hemangiomatosis with severe pulmonary hypertension. Circ J 67, 793–795. Jasmin, J. F., Lucas, M., Cernacek, P., & Dupuis, J. (2001). Effectiveness of a nonselective ET(A/B) and a selective ET(A) antagonist in rats with monocrotaline-induced pulmonary hypertension. Circulation 103, 314–318. Jeffery, T. K., & Wanstall, J. C. (2001). Pulmonary vascular remodeling: A target for therapeutic intervention in pulmonary hypertension. Pharmacol Ther 92, 1–20. Jiang, B. H., Tawara, S., Abe, K., Takaki, A., Fukumoto, Y., & Shimokawa, H. (2007). Acute vasodilator effect of fasudil, a Rho-kinase inhibitor, in monocrotaline-induced pulmonary hypertension in rats. J Cardiovasc Pharmacol 49, 85–89. Jones, R., Jacobson, M., & Steudel, W. (1999). alpha-smooth-muscle actin and microvascular precursor smooth-muscle cells in pulmonary hypertension. Am J Respir Cell Mol Biol 20, 582–594. Kamiyama, M., Utsunomiya, K., Taniguchi, K., Yokota, T., Kurata, H., Tajima, N., et al. (2003). Contribution of Rho A and Rho kinase to platelet-derived growth factor-BB-induced proliferation of vascular smooth muscle cells. J Atheroscler Thromb 10, 117–123. Kanda, T., Hayashi, K., Wakino, S., Homma, K., Yoshioka, K., Hasegawa, K., et al. (2005). Role of Rho-kinase and p27 in angiotensin II-induced vascular injury. Hypertension 45, 724–729.

Kawabata, S., Usukura, J., Morone, N., Ito, M., Iwamatsu, A., Kaibuchi, K., et al. (2004). Interaction of Rho-kinase with myosin II at stress fibres. Genes Cells 9, 653–660. Keil, A., Blom, I. E., Goldschmeding, R., & Rupprecht, H. D. (2002). Nitric oxide down-regulates connective tissue growth factor in rat mesangial cells. Kidney Int 62, 401–411. Khan, I., Oriowo, M. A., Chandrasekhar, B., & Kadavil, E. A. (2005). Attenuated noradrenaline-induced contraction of pulmonary arteries from rats treated with monocrotaline: Role of rho kinase. J Vasc Res 42, 433–440. Knock, G. A., Snetkov, V. A., Shaifta, Y., Connolly, M., Drndarski, S., Noah, A., et al. (2009). Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca(2+) sensitization. Free Radic Biol Med 46, 633–642. Koyama, M., Ito, M., Feng, J., Seko, T., Shiraki, K., Takase, K., et al. (2000). Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett 475, 197–200. Krepinsky, J. C., Ingram, A. J., Tang, D., Wu, D., Liu, L., & Scholey, J. W. (2003). Nitric oxide inhibits stretch-induced MAPK activation in mesangial cells through RhoA inactivation. J Am Soc Nephrol 14, 2790–2800. Lane, K. B., Machado, R. D., Pauciulo, M. W., Thomson, J. R., Phillips, J. A., III, Loyd, J. E., et al. (2000). Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. Nat Genet 26, 81–84. Laufs, U., Endres, M., & Liao, J. K. (1999a). Regulation of endothelial NO production by Rho GTPase. Med Klin (Munich) 94, 211–218. Laufs, U., & Liao, J. K. (1998). Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem 273, 24266–24271. Laufs, U., Marra, D., Node, K., & Liao, J. K. (1999b). 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1). J Biol Chem 274, 21926–21931. Leung, T., Chen, X. Q., Manser, E., & Lim, L. (1996). The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol 16, 5313–5327. Leung, T., Manser, E., Tan, L., & Lim, L. (1995). A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem 270, 29051–29054. Li, X. H., Peng, J., Tan, N., Wu, W. H., Li, T. T., Shi, R. Z., et al. (2010). Involvement of asymmetric dimethylarginine and Rho kinase in the vascular remodeling in monocrotaline-induced pulmonary hypertension. Vascul Pharmacol 53, 223–229. Li, F., Xia, W., Li, A., Zhao, C., & Sun, R. (2007). Long-term inhibition of Rho kinase with fasudil attenuates high flow induced pulmonary artery remodeling in rats. Pharmacol Res 55, 64–71. Li, F., Xia, W., Yuan, S., & Sun, R. (2009). Acute inhibition of Rho-kinase attenuates pulmonary hypertension in patients with congenital heart disease. Pediatr Cardiol 30, 363–366. Liao, J. K., & Laufs, U. (2005). Pleiotropic effects of statins. Annu Rev Pharmacol Toxicol 45, 89–118. Loirand, G., Guérin, P., & Pacaud, P. (2006). Rho kinases in cardiovascular physiology and pathophysiology. Circ Res 98, 322–334. Machado, R. D., Aldred, M. A., James, V., Harrison, R. E., Patel, B., Schwalbe, E. C., et al. (2006). Mutations of the TGF-beta type II receptor BMPR2 in pulmonary arterial hypertension. Hum Mutat 27, 121–132. Machado, R. D., Eickelberg, O., Elliott, C. G., Geraci, M. W., Hanaoka, M., Loyd, J. E., et al. (2009). Genetics and genomics of pulmonary arterial hypertension. J Am Coll Cardiol 54(1 Suppl.), S32–S42. Mahapatra, S., Nishimura, R. A., Oh, J. K., & McGoon, M. D. (2006a). The prognostic value of pulmonary vascular capacitance determined by Doppler echocardiography in patients with pulmonary arterial hypertension. J Am Soc Echocardiogr 19, 1045–1050. Mahapatra, S., Nishimura, R. A., Sorajja, P., Cha, S., & McGoon, M. D. (2006b). Relationship of pulmonary arterial capacitance and mortality in idiopathic pulmonary arterial hypertension. J Am Coll Cardiol 47, 799–803. Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., et al. (1996). Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J 15, 2208–2216. McNamara, P. J., Murthy, P., Kantores, C., Teixeira, L., Engelberts, D., van Vliet, T., et al. (2008). Acute vasodilator effects of Rho-kinase inhibitors in neonatal rats with pulmonary hypertension unresponsive to nitric oxide. Am J Physiol Lung Cell Mol Physiol 294, L205–L213. Moncada, S., Palmer, R. M., & Higgs, E. A. (1989). Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication. Biochem Pharmacol 38, 1709–1715. Montani, D., Price, L. C., Dorfmuller, P., Achouh, L., Jaïs, X., Yaïci, A., et al. (2009). Pulmonary veno-occlusive disease. Eur Respir J 33, 189–200. Moosmang, S., Schulla, V., Welling, A., Feil, R., Feil, S., Wegener, J. W., et al. (2003). Dominant role of smooth muscle L-type calcium channel Cav1.2 for blood pressure regulation. EMBO J 22, 6027–6034. Morgan, D. O. (1995). Principles of CDK regulation. Nature 374, 131–134. Morrell, N. W., Adnot, S., Archer, S. L., Dupuis, J., Jones, P. L., MacLean, M. R., et al. (2009). Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 54(1 Suppl.), S20–S31. Mouchaers, K. T. B., Schalij, I., de Boer, M. A., Postmus, P. E., van Hinsbergh, V. W. M., van Nieuw Amerongen, G. P., et al. (2010). Fasudil reduces monocrotaline-induced pulmonary arterial hypertension: Comparison with bosentan and sildenafil. Eur Respir J 36, 800–807. Naeije, R., & Huez, S. (2007). Expert opinion on available options treating pulmonary arterial hypertension. Expert Opin Pharmacother 8, 2247–2265. Nagaoka, T., Fagan, K. A., Gebb, S. A., Morris, K. G., Suzuki, T., Shimokawa, H., et al. (2005). Inhaled Rho kinase inhibitors are potent and selective vasodilators in rat pulmonary hypertension. Am J Respir Crit Care Med 171, 494–499.

S. Duong-Quy et al. / Pharmacology & Therapeutics 137 (2013) 352–364 Nagaoka, T., Gebb, S. A., Karoor, V., Homma, N., Morris, K. G., McMurtry, I. F., et al. (2006). Involvement of RhoA/Rho kinase signaling in pulmonary hypertension of the fawn-hooded rat. J Appl Physiol 100, 996–1002. Nagaoka, T., Morio, Y., Casanova, N., Bauer, N., Gebb, S., McMurtry, I., et al. (2004). Rho/Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 287, L665–L672. Nakagawa, O., Fujisawa, K., Ishizaki, T., Saito, Y., Nakao, K., & Narumiya, S. (1996). ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice. FEBS Lett 392, 189–193. Newman, J. H., Trembath, R. C., Morse, J. A., Grunig, E., Loyd, J. E., Adnot, S., et al. (2004). Genetic basis of pulmonary arterial hypertension: current understanding and future directions. J Am Coll Cardiol 43(12 Suppl. S), 33S–39S. Newman, J. H., Wheeler, L., Lane, K. B., Loyd, E., Gaddipati, R., Phillips, J. A., III, et al. (2001). Mutation in the gene for bone morphogenetic protein receptor II as a cause of primary pulmonary hypertension in a large kindred. N Engl J Med 345, 319–324. Ni, W., Egashira, K., Kataoka, C., Kitamoto, S., Koyanagi, M., Inoue, S., et al. (2001). Antiinflammatory and antiarteriosclerotic actions of HMG-CoA reductase inhibitors in a rat model of chronic inhibition of nitric oxide synthesis. Circ Res 89, 415–421. Nishimura, T., Vaszar, L. T., Faul, J. L., Zhao, G., Berry, G. J., Shi, L., et al. (2003). Simvastatin rescues rats from fatal pulmonary hypertension by inducing apoptosis of neointimal smooth muscle cells. Circulation 108, 1640–1645. Oka, M., Homma, N., Taraseviciene-Stewart, L., Morris, K. G., Kraskauskas, D., Burns, N., et al. (2007). Rho kinase-mediated vasoconstriction is important in severe occlusive pulmonary arterial hypertension in rats. Circ Res 100, 923–929. Olschewski, H., Simonneau, G., Galiè, N., Higenbottam, T., Naeije, R., Rubin, L. J., et al. (2002). Inhaled iloprost for severe pulmonary hypertension. N Engl J Med 347, 322–329. Palevsky, H. I., Schloo, B. L., Pietra, G. G., Weber, K. T., Janicki, J. S., Rubin, E., et al. (1989). Primary pulmonary hypertension. Vascular structure, morphometry, and responsiveness to vasodilator agents. Circulation 80, 1207–1221. Perros, F., Dorfmüller, P., Souza, R., Durand-Gasselin, I., Godot, V., Capel, F., et al. (2007a). Fractalkine-induced smooth muscle cell proliferation in pulmonary hypertension. Eur Respir J 29, 937–943. Perros, F., Dorfmüller, P., Souza, R., Durand-Gasselin, I., Mussot, S., Mazmanian, M., et al. (2007b). Dendritic cell recruitment in lesions of human and experimental pulmonary hypertension. Eur Respir J 29, 462–468. Piddini, E., Schmid, J. A., de Martin, R., & Dotti, C. G. (2001). The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9. EMBO J 20, 4076–4087. Pierson, D. J. (2000). Pathophysiology and clinical effects of chronic hypoxia. Respir Care 45, 39–51 (discussion 51–53). Pietra, G. G., Capron, F., Stewart, S., Leone, O., Humbert, M., Robbins, I. M., et al. (2004). Pathologic assessment of vasculopathies in pulmonary hypertension. J Am Coll Cardiol 43(12 Suppl. S), 25S–32S. Rabinovitch, M., Gamble, W., Nadas, A. S., Miettinen, O. S., & Reid, L. (1979). Rat pulmonary circulation after chronic hypoxia: Hemodynamic and structural features. Am J Physiol 236, H818–H827. Riento, K., Guasch, R. M., Garg, R., Jin, B., & Ridley, A. J. (2003). RhoE binds to ROCK I and inhibits downstream signaling. Mol Cell Biol 23, 4219–4229. Riento, K., & Ridley, A. J. (2003). Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 4, 446–456. Rikitake, Y., Kim, H. -H., Huang, Z., Seto, M., Yano, K., Asano, T., et al. (2005). Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke 36, 2251–2257. Rikitake, Y., & Liao, J. K. (2005). Rho GTPases, statins, and nitric oxide. Circ Res 97, 1232–1235. Rubin, L. J., Badesch, D. B., Barst, R. J., Galie, N., Black, C. M., Keogh, A., et al. (2002). Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 346, 896–903. Sakao, S., Taraseviciene-Stewart, L., Cool, C. D., Tada, Y., Kasahara, Y., Kurosu, K., et al. (2007). VEGF-R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle-like and neuronal-like cells. FASEB J 21, 3640–3652. Sakurada, S., Okamoto, H., Takuwa, N., Sugimoto, N., & Takuwa, Y. (2001). Rho activation in excitatory agonist-stimulated vascular smooth muscle. Am J Physiol Cell Physiol 281, C571–C578. Sanchez, O., Marcos, E., Perros, F., Fadel, E., Tu, L., Humbert, M., et al. (2007). Role of endothelium-derived CC chemokine ligand 2 in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 176, 1041–1047. Sastry, B. K. S., Narasimhan, C., Reddy, N. K., & Raju, B. S. (2004). Clinical efficacy of sildenafil in primary pulmonary hypertension: A randomized, placebo-controlled, double-blind, crossover study. J Am Coll Cardiol 43, 1149–1153. Sata, M. (2006). Role of circulating vascular progenitors in angiogenesis, vascular healing, and pulmonary hypertension: Lessons from animal models. Arterioscler Thromb Vasc Biol 26, 1008–1014. Satoh, S., Utsunomiya, T., Tsurui, K., Kobayashi, T., Ikegaki, I., Sasaki, Y., et al. (2001). Pharmacological profile of hydroxy fasudil as a selective rho kinase inhibitor on ischemic brain damage. Life Sci 69, 1441–1453. Sauzeau, V., Le Jeune, H., Cario-Toumaniantz, C., Smolenski, A., Lohmann, S. M., Bertoglio, J., et al. (2000). Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem 275, 21722–21729. Sauzeau, V., Le Mellionnec, E., Bertoglio, J., Scalbert, E., Pacaud, P., & Loirand, G. (2001). Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase. Circ Res 88, 1102–1104.

363

Sawada, N., Itoh, H., Ueyama, K., Yamashita, J., Doi, K., Chun, T. H., et al. (2000). Inhibition of rho-associated kinase results in suppression of neointimal formation of balloon-injured arteries. Circulation 101, 2030–2033. Seasholtz, T. M., Majumdar, M., Kaplan, D. D., & Brown, J. H. (1999). Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration. Circ Res 84, 1186–1193. Sebbagh, M., Hamelin, J., Bertoglio, J., Solary, E., & Bréard, J. (2005). Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner. J Exp Med 201, 465–471. Sebbagh, M., Renvoizé, C., Hamelin, J., Riché, N., Bertoglio, J., & Bréard, J. (2001). Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol 3, 346–352. Sherr, C. J., & Roberts, J. M. (1995). Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9, 1149–1163. Shimokawa, H. (2002). Rho-kinase as a novel therapeutic target in treatment of cardiovascular diseases. J Cardiovasc Pharmacol 39, 319–327. Shimokawa, H., Hiramori, K., Iinuma, H., Hosoda, S., Kishida, H., Osada, H., et al. (2002). Anti-anginal effect of fasudil, a Rho-kinase inhibitor, in patients with stable effort angina: A multicenter study. J Cardiovasc Pharmacol 40, 751–761. Shimokawa, H., Seto, M., Katsumata, N., Amano, M., Kozai, T., Yamawaki, T., et al. (1999). Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm. Cardiovasc Res 43, 1029–1039. Shirao, S., Kashiwagi, S., Sato, M., Miwa, S., Nakao, F., Kurokawa, T., et al. (2002). Sphingosylphosphorylcholine is a novel messenger for Rho-kinase-mediated Ca2+ sensitization in the bovine cerebral artery: unimportant role for protein kinase C. Circ Res 91, 112–119. Simonneau, G., Galiè, N., Rubin, L. J., Langleben, D., Seeger, W., Domenighetti, G., et al. (2004). Clinical classification of pulmonary hypertension. J Am Coll Cardiol 43(12 Suppl. S), 5S–12S. Sin, W. C., Chen, X. Q., Leung, T., & Lim, L. (1998). RhoA-binding kinase alpha translocation is facilitated by the collapse of the vimentin intermediate filament network. Mol Cell Biol 18, 6325–6339. Sitbon, O., Humbert, M., Nunes, H., Parent, F., Garcia, G., Hervé, P., et al. (2002). Long-term intravenous epoprostenol infusion in primary pulmonary hypertension: prognostic factors and survival. J Am Coll Cardiol 40, 780–788. Somlyo, A. P., & Somlyo, A. V. (2003). Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: Modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 83, 1325–1358. Sommer, N., Dietrich, A., Schermuly, R. T., Ghofrani, H. A., Gudermann, T., Schulz, R., et al. (2008). Regulation of hypoxic pulmonary vasoconstriction: Basic mechanisms. Eur Respir J 32, 1639–1651. Stamler, J. S., Lamas, S., & Fang, F. C. (2001). Nitrosylation. The prototypic redox-based signaling mechanism. Cell 106, 675–683. Steiner, M. K., Syrkina, O. L., Kolliputi, N., Mark, E. J., Hales, C. A., & Waxman, A. B. (2009). Interleukin-6 overexpression induces pulmonary hypertension. Circ Res 104, 236–244 (28 pp. following 244). Sztrymf, B., Coulet, F., Girerd, B., Yaici, A., Jais, X., Sitbon, O., et al. (2008). Clinical outcomes of pulmonary arterial hypertension in carriers of BMPR2 mutation. Am J Respir Crit Care Med 177, 1377–1383. Takahashi, N., Tuiki, H., Saya, H., & Kaibuchi, K. (1999). Localization of the gene coding for ROCK II/Rho kinase on human chromosome 2p24. Genomics 55, 235–237. Takemoto, M., Sun, J., Hiroki, J., Shimokawa, H., & Liao, J. K. (2002). Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation 106, 57–62. Takizawa, N., Koga, Y., & Ikebe, M. (2002). Phosphorylation of CPI17 and myosin binding subunit of type 1 protein phosphatase by p21-activated kinase. Biochem Biophys Res Commun 297, 773–778. Tamby, M. C., Chanseaud, Y., Humbert, M., Fermanian, J., Guilpain, P., Garciade-la-Peña-Lefebvre, P., et al. (2005). Anti-endothelial cell antibodies in idiopathic and systemic sclerosis associated pulmonary arterial hypertension. Thorax 60, 765–772. Tawara, S., Fukumoto, Y., & Shimokawa, H. (2007). Effects of combined therapy with a Rho-kinase inhibitor and prostacyclin on monocrotaline-induced pulmonary hypertension in rats. J Cardiovasc Pharmacol 50, 195–200. Toshima, Y., Satoh, S., Ikegaki, I., & Asano, T. (2000). A new model of cerebral microthrombosis in rats and the neuroprotective effect of a Rho-kinase inhibitor. Stroke 31, 2245–2250. Trembath, R. C., Thomson, J. R., Machado, R. D., Morgan, N. V., Atkinson, C., Winship, I., et al. (2001). Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N Engl J Med 345, 325–334. Tuder, R. M., Abman, S. H., Braun, T., Capron, F., Stevens, T., Thistlethwaite, P. A., et al. (2009). Development and pathology of pulmonary hypertension. J Am Coll Cardiol 54(1 Suppl.), S3–S9. Tuder, R. M., Cool, C. D., Geraci, M. W., Wang, J., Abman, S. H., Wright, L., et al. (1999). Prostacyclin synthase expression is decreased in lungs from patients with severe pulmonary hypertension. Am J Respir Crit Care Med 159, 1925–1932. Tuder, R. M., Groves, B., Badesch, D. B., & Voelkel, N. F. (1994). Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension. Am J Pathol 144, 275–285. Tuder, R. M., & Voelkel, N. F. (1998). Pulmonary hypertension and inflammation. J Lab Clin Med 132, 16–24. Tuder, R. M., & Zaiman, A. L. (2004). Pathology of pulmonary vascular disease. In A. Peacock, & L. J. Rubin (Eds.), Pulmonary circulation (pp. 25–32). (2nd ed.). London: Arnold. Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T., et al. (1997). Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389, 990–994.

364

S. Duong-Quy et al. / Pharmacology & Therapeutics 137 (2013) 352–364

VanBavel, E., Sorop, O., Andreasen, D., Pfaffendorf, M., & Jensen, B. L. (2002). Role of T-type calcium channels in myogenic tone of skeletal muscle resistance arteries. Am J Physiol Heart Circ Physiol 283, H2239–H2243. Vanderpool, R. R., Kim, A. R., Molthen, R., & Chesler, N. C. (2011). Effects of acute Rho kinase inhibition on chronic hypoxia-induced changes in proximal and distal pulmonary arterial structure and function. J Appl Physiol 110, 188–198. Velasco, G., Armstrong, C., Morrice, N., Frame, S., & Cohen, P. (2002). Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin. FEBS Lett 527, 101–104. Voelkel, N. F., Cool, C., Lee, S. D., Wright, L., Geraci, M. W., & Tuder, R. M. (1998). Primary pulmonary hypertension between inflammation and cancer. Chest 114(3 Suppl.), 225S–230S. Wagenvoort, C. A., & Wagenvoort, N. (1970). Primary pulmonary hypertension a pathologic study of the lung vessels in 156 clinically diagnosed cases. Circulation 42, 1163–1184. Ward, Y., Yap, S. F., Ravichandran, V., Matsumura, F., Ito, M., Spinelli, B., et al. (2002). The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway. J Cell Biol 157, 291–302. Wibberley, A., Chen, Z., Hu, E., Hieble, J. P., & Westfall, T. D. (2003). Expression and functional role of Rho-kinase in rat urinary bladder smooth muscle. Br J Pharmacol 138, 757–766. Wilson, D. W., Segall, H. J., Pan, L. C., & Dunston, S. K. (1989). Progressive inflammatory and structural changes in the pulmonary vasculature of monocrotaline-treated rats. Microvasc Res 38, 57–80. Wilson, D. P., Susnjar, M., Kiss, E., Sutherland, C., & Walsh, M. P. (2005). Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2+ entry and Ca2+ sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855, but not Thr-697. Biochem J 389, 763–774. Wilson, D. P., Sutherland, C., & Walsh, M. P. (2002). Ca2+ activation of smooth muscle contraction: evidence for the involvement of calmodulin that is bound to the triton insoluble fraction even in the absence of Ca2+. J Biol Chem 277, 2186–2192.

Wolfrum, S., Dendorfer, A., Rikitake, Y., Stalker, T. J., Gong, Y., Scalia, R., et al. (2004). Inhibition of Rho-kinase leads to rapid activation of phosphatidylinositol 3-kinase/protein kinase Akt and cardiovascular protection. Arterioscler Thromb Vasc Biol 24, 1842–1847. Xu, E. Z., Kantores, C., Ivanovska, J., Engelberts, D., Kavanagh, B. P., McNamara, P. J., et al. (2010). Rescue treatment with a Rho-kinase inhibitor normalizes right ventricular function and reverses remodeling in juvenile rats with chronic pulmonary hypertension. Am J Physiol Heart Circ Physiol 299, H1854–H1864. Yamakawa, T., Tanaka, S., Numaguchi, K., Yamakawa, Y., Motley, E. D., Ichihara, S., et al. (2000). Involvement of Rho-kinase in angiotensin II-induced hypertrophy of rat vascular smooth muscle cells. Hypertension 35, 313–318. Yi, E. S., Kim, H., Ahn, H., Strother, J., Morris, T., Masliah, E., et al. (2000). Distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension. A morphometric and immunohistochemical study. Am J Respir Crit Care Med 162, 1577–1586. Yuan, J. X., Aldinger, A. M., Juhaszova, M., Wang, J., Conte, J. V., Jr., Gaine, S. P., et al. (1998). Dysfunctional voltage-gated K+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation 98, 1400–1406. Yuan, J. X. J., & Rubin, L. J. (2005). Pathogenesis of pulmonary arterial hypertension: the need for multiple hits. Circulation 111, 534–538. Zhu, P., Huang, L., Ge, X., Yan, F., Wu, R., & Ao, Q. (2006). Transdifferentiation of pulmonary arteriolar endothelial cells into smooth muscle-like cells regulated by myocardin involved in hypoxia-induced pulmonary vascular remodelling. Int J Exp Pathol 87, 463–474. Ziino, A. J. A., Ivanovska, J., Belcastro, R., Kantores, C., Xu, E. Z., Lau, M., et al. (2010). Effects of rho-kinase inhibition on pulmonary hypertension, lung growth, and structure in neonatal rats chronically exposed to hypoxia. Pediatr Res 67, 177–182. Zuckerbraun, B. S., Stoyanovsky, D. A., Sengupta, R., Shapiro, R. A., Ozanich, B. A., Rao, J., et al. (2007). Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA. Am J Physiol Cell Physiol 292, C824–C831.