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Pharmacology & Therapeutics
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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, Franceb Clinical and Translational Research Center, Tongji University School of Medicine and Shanghai East Hospital, 150 Jimo Road, Shanghai, 200120, China
Pulmonary hypertension (PH) is an incurable disease with a dreadful survival rate. The disease is characterizedby sustained vasoconstriction, progressive vascular remodeling, and irreversible right heart dysfunction. Whilehypoxic pulmonary vasoconstriction (HPV) is known to be the main pathophysiological factor causing the risein pulmonary arterial pressure, biological mechanisms leading to HPV and vascular remodeling are multipleand complex and, as yet, incompletely understood. It is thought that molecular interactions and cross talks areinvolved in the pathogenesis of PH, perturbing the physiological balance between substances controlling vascu-lar tone, cell growth and apoptosis. This balance is achieved by subtle interactions between factors acting as bothvasodilators and inhibitors of cell growth like nitric oxide, prostacyclin, vasoactive intestinal peptide and mole-culeswith potent vasoconstrictor and cell growth activities like endothelin-1. Recent in vivo studies showed thatthe Rho GTPase/RhoA pathway and its downstream effectors, the Rho-kinases (ROCK-1 and ROCK-2), had an im-portant role in PH, due to its lasting effects on vasoconstriction and pulmonary cell proliferation leading to vas-cular remodeling. Beneficial effects obtained in vivo with Rho-kinase inhibitors (e.g.Y-27632 and fasudil) inexperimental PHwill hopefully lead to future clinical trials with new compounds selectively targeting this path-way, which is now proven to be detrimental when over-activated in both experimental animals and humanpatients.
Pulmonary hypertension (PH) is a rare disorder with a dreadful prog-nosis. Despite substantial advances in the knowledge of pathogenesis andtherapy during the last decade, PH still remains an incurable disease. Inthe 90s, the median survival rate of untreated patients was around2.8 years from diagnosis (D'Alonzo et al., 1991). PH is defined by an in-crease in mean pulmonary arterial pressure (PAP) ≥25 mm Hg at rest,a pulmonary wedge pressure (PWP) ≤15 mm Hg and a normal or re-duced cardiac output, assessed by right heart catheterization (Galiè etal., 2009). PH is currently classified into five groups and one subgroupaccording to pathological, clinical, and therapeutic features (Galiè et al.,2009).
Current treatment of PH consists of the use of conventional therapy incombination with specific treatments with continuous prostacyclin infu-sion or inhalation, oral phosphodiesterase-5 inhibitors (Olschewski etal., 2002; Sitbon et al., 2002; Sastry et al., 2004) and oral endothelin-1 re-ceptor antagonists (Channick et al., 2001; Rubin et al., 2002; Naeije &Huez, 2007). None of these drugs, however, can be considered as an opti-mal treatment for PH as theymainly act as vasodilators and lack inhibito-ry effects on remodeling of the pulmonary vasculature. There is thereforea need for new treatments to be found and tested by randomized clinicaltrials (Hoeper & Dinh-Xuan, 2004).
Recently, accumulating evidence showed that RhoA and its down-stream effectors, the Rho-kinases, have a preponderant role in the phys-iopathology of PH due to their potent effects on pulmonary arterialsmooth muscle cell (SMCs) contraction and proliferation (Nagaoka etal., 2006; Homma et al., 2008; Guilluy et al., 2009). This paperwill reviewthe biological role of Rho-kinase signaling pathway and the efficacy ofRho-kinase inhibitors in the treatment of PH, targeting both its vasocon-strictor 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 theWorld Health Organization (WHO) Symposium held in Evian, catego-rized the disorder as “primary”whenno underlying etiology or risk factorcould be identified, and “secondary”when clearly associated to a clearlyidentified primary disorder (Hatano & Strasser, 1975). Since then, theclinical classification of PH has been revised thrice in 1998 (Evian,France), 2003 (Venice, Italy) (Simonneau et al., 2004), and 2008 (DanaPoint, USA) (Galiè et al., 2009). In these classifications, clinical conditionswith PH were divided into five groups according to pathological, patho-physiological, and therapeutic characteristics.
The latest clinical classification, made in Dana Point in 2008(Galiè et al., 2009) maintains the overall structure of the Evian-Venice classifications while amending some specific points toavoid possible confusion among the terms PH and PAH (pulmonaryarterial hypertension). This new classification of PH is summarizedas follows:
Group 1 (PAH): including idiopathic PAH, heritable PAH (germlinemutations of BMPR2, ALK1 or endoglin genes), drugs and toxins in-duced PAH, PAH associated with other conditions (connective tissuediseases, HIV infection, portal hypertension, congenital heart disease,schistosomiasis, chronic hemolytic anemia), and persistent pulmo-nary hypertension of the newborn.Group1′: PH due to pulmonary veno-occlusive disease and/orpulmonary capillary hemangiomatosis. These conditions havebeen classified as a distinct category but not completely separat-ed from PAH, and designated as Group 1′.Group 2: PH related to left heart disease, including systolic dys-function, diastolic dysfunction, and valvular disorders.
Group 3: PH related to lung disease and/or hypoxia, including chron-ic obstructive pulmonary disease, interstitial lung disease, other pul-monary 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 pre-cise criterion to distinguish between proximal and distal forms.Group 5: PH with unclear and/or multifactorial mechanisms, includ-ing heterogeneous conditions with different pathological featuressuch as hematological disorders (myeloproliferative disorders, sple-nectomy), systemic disorders (sarcoidosis, pulmonary Langerhanscell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis,vasculitis), metabolic disorders (glycogen storage disease, Gaucherdisease, thyroid disorders), and others (tumoral obstruction, fibrosingmediastinitis, chronic renal failure on dialysis).
2.2. Physiopathology and pathogenesis of pulmonary hypertension
Pulmonary vasoconstriction is an important feature of all forms ofPH. Decrease of alveolar partial pressure of oxygen results in acutepulmonary vasoconstriction which, when prolonged, will eventuallylead to the occurrence of PH (Sommer et al., 2008). Chronic hypoxicpulmonary vasoconstriction is sustained and worsened by an imbal-ance between vasodilators and vasoconstrictors acting on the pulmo-nary vascular smooth muscle cells. Recent evidence also suggestsdeleterious roles of growth factors (Giaid & Saleh, 1995; Tuder et al.,1999; Galié et al., 2004). Other mechanisms involved in the physiopa-thology of PH include 1/reduced cross-sectional area of pulmonary vas-cular bed due to disorders affecting the lung parenchyma, 2/thrombiobstructing pulmonary vascular lumen (Mahapatra et al., 2006a,2006b), 3/overloaded volume and pressure caused by left-right intra-cardiac shunts, 4/obstruction of pulmonary venousflow, and 5/a combi-nation 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 dependson clinical features, the pathogenesis of PH is complex and incom-pletely understood. It is thought that the underlying mechanismsare multiple and lead to both functional and structural changes ofthe pulmonary vasculature (Humbert et al., 2004; Yuan & Rubin,2005).
2.3. Pathology and pathobiology of pulmonary hypertension
Although differentmechanisms are involved in the physiopathologyand pathogenesis of the disease, all forms of PH have common patho-logic features in pulmonary arteries. It is mostly characterized by vascu-lar remodeling (medial hypertrophy, intimal fibrosis, and adventitialthickening), and organized thrombotic (Pietra et al., 2004; Yuan &Rubin, 2005; Tuder et al., 2009). Pulmonary vascular remodeling ischaracterized by the increasing of external diameter of pulmonary ar-teries due to predominantly SMC hypertrophy and extracellular matrixand collagen deposit (Palevsky et al., 1989; Yi et al., 2000). However, thecorrelation betweenmorphological 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 andmultifactorial (Humbertet al., 2004; Hassoun et al., 2009; Morrell et al., 2009) (Fig. 1). Manymediator and signaling pathway with its downstream effectors areimplicated in the pathobiology of PH. It is involved in the regulation ofpulmonary vasoactivity, endothelial function, SMC proliferation, andvascular inflammation or thrombosis. Impairment of vasoconstriction–vasodilatation balance is the earliest disorder occurring in the pathogen-esis of PAH (Christman et al., 1992; Giaid & Saleh, 1995). Endothelial
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 cAMPwhereas 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 triphos-phate) 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 isregulated 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-JunN-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, vasoactiveintestinal peptide.
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dysfunction leads to chronically impaired production of vasodilator andanti-proliferative agents such as NO and prostacyclin, along withoverexpression of vasoconstrictor and proliferative substances such asthromboxane A2 and endothelin-1 (ET-1). Inflammatory cells, platelets(through the serotonin pathway), andprothrombotic abnormalities alsoplay a significant role in pathogenesis of PH.
However, the role of other molecular and signaling pathway, whichismediated bymany factors such as ET-1, serotonin (5-HT), angiotensinII (Ang II), growth factor etc.…, in pathobiology and pathogenesis of PHis not clearly clarified.
2.4. Genetic disorders in pulmonary hypertension
The best characterized genetic defects in heritable pulmonaryarterial hypertension are mutations of the gene encoding bonemorphogenetic protein receptor type 2 (BMPR2), a member of thetransforming growth factor-β signaling family. BMPR2 modulatesthe growth of vascular cells by activating the intracellular pathwayof Smad and LIM kinase (Foletta et al., 2003). The germline muta-tions 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 ap-parently sporadic cases, thus representing the major geneticpredisposing factor for PAH (Sztrymf et al., 2008). More than 45 dif-ferent mutations of BMPR2 gene have been identified in patientswith heritable PAH (Lane et al., 2000; Newman et al., 2001). Func-tional studies have shown that point mutations and truncations in thekinase domain exert dominant negative effects on receptor function(Newman et al., 2004), resulting in incomplete penetrance and geneticanticipation.
Mutations of other receptors such as activin receptor-like kinase 1(ALK1) and endoglin have also been identified in PAH patients usuallyfrom families with coexistent hereditary hemorrhagic telangiectasia(Trembath et al., 2001). Mutations in ALK1 are believed to result incellular growth-promoting via Smad-depending signaling. Geneticmutations of the serotonin transporter (5-HTT) are more frequentin idiopathic PAH than control subjects (Eddahibi et al., 2001). TheL-allelic variant of the 5HTT gene is associated with an increased ex-pression of the transporter and increased proliferation of vascularSMC. Serotonin gene polymorphism has also been found in PH
patients with hypoxemic chronic obstructive pulmonary disease(COPD) (Eddahibi et al., 2003).
2.5. Role of inflammation
The roles of inflammation and autoimmunity in vascular remod-eling seen in idiopathic PH and PH associated with systemic diseaseshave been recently highlighted (Tuder & Voelkel, 1998; Voelkel et al.,1998). First, circulating autoantibodies directed against endothelialcells and cell nuclei are frequently found in PH (Isern et al., 1992;Dorfmüller et al., 2003; Tamby et al., 2005). Secondly, perivascularinfiltration of inflammatory cells including lymphocytes (T and B),macrophages, and dendritic cells, is a constant feature of plexiformvascular lesions (Tuder et al., 1994; Pietra et al., 2004; Perros et al.,2007b).
Cytokines and inflammatory chemokines are also involved in thepathogenesis of PH. Increased concentrations and expressions ofpro-inflammatory cytokines (IL-1β, IL-6) were found in plasma andlung tissue of patients with severe idiopathic PAH (Humbert et al.,1995). In mice, high expression of IL-6 was associated with increasedpulmonary vascular resistance, and extensive pulmonary vascularlesions (Steiner et al., 2009). Efficacy of tocilizumab, a monoclonal an-tibody directed against IL-6 receptors, has been recently reported in apatient with PH (Furuya et al., 2010). Plasma concentrations and ex-pressions of fractalkine and its receptors (CX3CR1 and CX3CL1) areincreased in circulating lymphocytes (CD4+ and CD8+) and lung tis-sue (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 ofPH. It is due to encroach of vascular walls into the lumen, thus reducingthe vessels inner diameter. Many cell types of pulmonary vessel and cir-culation contribute to the development of pulmonary vascular remod-eling. This remodeling is characterized by the thickening of all thethree layers of pulmonary vessels, including the media, the intima,and the adventitia. It is marked by the hypertrophy and hyperplasia ofdifferent cell types such as endothelial cells (EC), smooth muscle cells(SMC), and fibroblasts. These structural changes are enhanced by theaccumulation of extracellular matrix components, including collagen,elastin, fibronectin, and tenascin, in the vascular walls, and predomi-nantly in the adventitia (Jeffery & Wanstall, 2001).
In the remodeled of pulmonary vessels, the thickened andhypertrophied media layer critically reduces vascular lumen whilstperturbing the fragile balance between vascular contraction andrelaxation mediators. Medial hypertrophy results from imbalancebetween proliferation and apoptosis of SMC, occurring at all levelsof pulmonary vascular tree, from large arteries to small arterioles(Dingemans & Wagenvoort, 1978). Furthermore, medial hypertro-phy further obstructs vascular lumen through extension of newmuscle cells into muscular and non muscular pulmonary arterioles,termed as the muscularization (Wagenvoort & Wagenvoort, 1970).Muscularization of pulmonary vessels is initiated by the differentia-tion of many precursor cells and fibroblasts into SMC (Jones et al.,1999; Sata, 2006).
Pulmonary vascular remodeling is also characterized by hypertrophyof the intima. Lung injury caused by hypoxia, inflammation, and shearstress, results in endothelial cells damage and dysfunction, uncontrolledcell proliferation, excessive vasoconstriction, and in situ thrombosis.These processes are linked to complex patterns of inflammation, angio-genesis, and trans-differentiation of endothelial cells to pulmonary vas-cular SMC (Zhu et al., 2006; Sakao et al., 2007), leading to intimalproliferation and lamina-intima fibrosis and yielding plexiform lesionsof pulmonary arteries.
3. Rho-kinase signaling pathway
3.1. Molecular structure and expression of Rho-kinase
Rho-kinase, also namedRho-associated kinase and identified inmild1990s, is one of the effectors of Rho families (Ishizaki et al., 1996).Rho-kinase or ROCKs having two different isoforms, ROCK-1or ROCK-β and ROCK-2 or ROCK-α, is the main downstream effectors ofGTPase-RhoA (Ishizaki et al., 1996). ROCKs are serine/threoninekinases with a molecular mass of ~160 kD. These kinases are formedby parallel homodimers including a catalytic (kinase) domain in itsamino-terminus (NH2- or N-terminal domain), a coiled-coil in itsmiddle dimerization portion, and a putative Pleckstrin-homology(PH) domain in its cystein-riche domain (COOH- or C-terminaldomain, CRD) (Fig. 2) (Ishizaki et al., 1996; Matsui et al., 1996;Fukata et al., 2001). These carboxyl terminal domains constitute anautoinhibitory region that reduces the kinase activity of ROCKs(Amano et al., 1999). The Rho-binding domain (RBD) of ROCKs is lo-calized in the C-terminal portion of the coiled-coil region, and itshows sequence homology to the Rho-interaction domain of kinectinwhich is a regulating protein of microtubule-based organelle motility(Alberts et al., 1998) (Fig. 2). The coiled-coil region of ROCKs is showedto interact with other α-helical proteins, whereas the PH domain is in-volved 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 geneslocalizing on chromosome 18 (18q11.1) and chromosome 2 (2p24),respectively (Nakagawa et al., 1996; Takahashi et al., 1999). Twoisoformes of Rho-kinase in human have the homologue structurewith about 65% of amino acid and 58% of RBD. The highest similarity(92%) is presented at kinase domain (Leung et al., 1995; Nakagawa etal., 1996). The mRNAs of ROCK-1 and ROCK-2 are ubiquitouslyexpressed, with a preferential expression of ROCK-2 mRNA in brainand skeletal muscle. Both ROCK-1 and ROCK-2 are expressed in vas-cular smooth muscle and in heart (Leung et al., 1996; Wibberley etal., 2003). Cell-fractionation studies show that ROCKs are mainly dis-tributed in the cytoplasm fraction but a small amount of ROCKs is alsofound 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 subcellularlocalization at the vimentin intermediate-filament network and atactin 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 andPH domain) plays a role as a dominant-negative autoinhibitor (Fig. 3)due to its independent interaction with the catalytic domain (N-termi-nus) (Amano et al., 1999). Lacking of the C-terminus of ROCKs (truncat-ed forms) are constitutively formed the active form kinase (Amano etal., 2000). Beside of the self-associative autoinhibition, the activity ofROCKs is also influenced by its affinity for ATP which is regulated bythe dimerization of kinases (Doran et al., 2004).
Binding of GTPase-RhoA (amino acids 23–40 and 75–119 ofRhoA) to ROCKs at Rho-binding domain induces conformationalchanges of ROCKs, resulting in relieve of autoinhibitory blockageof kinase activity (Fujisawa et al., 1998). This binding is believedto stimulate the phosphotransferase activity of ROCKs (positive reg-ulation) by disrupting the interaction between catalytic and theC-terminal region of proteins (Loirand et al., 2006), which therebyfrees the kinase activity (Fig. 4a).
Independently of RhoA, ROCK activity might be activated by otherstimulators such as arachidonic acid (AA), sphingosine phosphoryl-choline (SPC), caspase-3 or granzyme B (Feng et al., 1999b; Shirao etal., 2002; Sebbagh et al., 2001, 2005). AA and SPC interact with thenegative regulatory region at PH domain, thus disrupting its inhibitory
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-bindingdomain (RBD). The Pleckstrin-homology (PH) with an internal cystein-rich domain (CRD) is situated in the carboxyl terminus (C-terminus).
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property on the catalytic activity of ROCKs (Feng et al., 1999b; Shirao etal., 2002). Caspase-3 cleaves ROCK-1 at the cleavage site DETD1113whereas 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 smallG-binding proteins such as RhoE, Gem, and Rad (Ward et al., 2002;Riento et al., 2003). RhoE binds to the N-terminal region of kinasedomain of ROCK-1 (amino acids 1–420) and therefore interfereswith the kinase activity and prevents GTPase-RhoA binding toRho-binding domain (Fig. 4b) (Riento et al., 2003). Overexpressionof Gem and Rad might inhibit respectively the downstream re-sponses of ROCK-1 and ROCK-2, but the mechanism is not clearlydemonstrated (Ward et al., 2002). In addition, the negative regula-tion of ROCK-mediated target effects by these small G-binding pro-teins are localized at the different cellular structure (RhoE in theGolgi and Gem and Rad in the cytoskeleton) (Bilan et al., 1998;Piddini et al., 2001).
4. Vascular downstream effect of Rho-kinase
4.1. Rho-kinase and vascular smooth muscle cells (VSMC)
4.1.1. Rho-kinase and vascular smooth muscle cells contractilityVascular smooth muscle cell (VSMC) contractility is dependent on
the level of phosphorylation of the 20 kDamyosin light chain (MLC20)that is determined by both Ca2+-dependent and Ca2+-independentmechanisms (Fig. 5).
VSMC Ca2+-dependent contractility is related to the concentrationof Ca2+ in cytosolic ([Ca2+]i) which is regulated by the release ofCa2+ from the sarcoplasmic reticulum and the entry of Ca2+ from theextracellular space via voltage-dependent Ca2+ channels (VDCCs) orthrough non-selective cation channels (NSCCs) (VanBavel et al., 2002;Moosmang et al., 2003). Increase of [Ca2+]i in cytosolic promotes there-fore the binding of Ca2+ to a specific pool of calmodulin (CaM) tetheredtomyosin light chain kinase (MLCK) (Wilson et al., 2002). It results thento the activation of MLCK and MLCK-mediated phosphorylation ofMLC20, thereby activating cross-bridge cycling and contraction.
Fig. 3. The autoinhibition form of ROCKs. The Pleckstrin-homology (PH) and Rho-binding
Vascular Ca2+-independent contractivity is related to Ca2+ sensitiza-tion of VSMC that is dependent on an increase of MLC20 phosphorylationand a force of contractile myofilaments without eliciting a change in[Ca2+]i. Many cellular signaling pathways contribute to culminate the in-hibition of myosin light chain phosphatase (MLCP) activity, increasingtherefore the level of phosphorylated MLC20 (Somlyo & Somlyo, 2003).ROCKs, downstream effectors of GTPase-RhoA, phosphorylate theMLCP targeting subunit (MYPT1) at threonine (Thr)-696, Thr-853,and Thr-855 (Somlyo & Somlyo, 2003; Wilson et al., 2005; Knock etal., 2009), resulting in inhibition of MLCP activity and increasing ofMLC20 phosphorylation (Feng et al., 1999a; Velasco et al., 2002).This in turn, enhances the actin binding and actin-induced ATPaseactivity of myosin, facilitating interaction of myosin with F-actin,then VSMC contractility (Fig. 5).
However, the inhibition of MLCP activity might bemediated by a di-rect interaction of the small 17 kDa protein kinase C-potentiated myo-sin phosphatase inhibitor protein (CPI-17) with protein phosphatase 1catalytic subunit (PP1c) of MLCP. Phosphorylation of CPI-17 by ROCKsat 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.
4.1.2. Rho-kinase and vascular smooth muscle cells proliferationThe 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 progressionthrough the G1 phase is regulated by phosphorylation and inactivationof 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, neg-atively regulated by the Cip/Kip family (p21Cip1, p27Kip1, and p57Kip2)of Cyclin-dependent kinase inhibitors (CDKIs) (Morgan, 1995; Sherr &Roberts, 1995). Hence, the cyclin-dependent kinase inhibitor p27Kip1
plays a crucial role in VSMC proliferation.There is increasing evidence that ROCK effectors downregulate
p27Kip1 expression, leading to the acceleration of cell cycle progressionand VSMC proliferation (Laufs et al., 1999b; Sawada et al., 2000). Theeffect of ROCK inhibitors as antiproliferators has been also ascribed to
domain (RBD) bind to the N-terminus of the enzyme, forming an autoinhibitory loop.
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. Ar-achidonic acid (AA) and sphingosine phosphorylcholine (SPC) interact with the negative regulatory region at PH domain, disrupting the inhibitory property of ROCKs. Caspase-3and 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 ofkinase domain of ROCKs (ROCK-1) prevents GTPase-RhoA binding to Rho-binding domain (RBD).
the upregulation of p27Kip1 expression (Sawada et al., 2000; Kanda etal., 2005). In addition, the role of ROCKs in the proliferation of VSMCme-diated by platelet-derived growth factor (PDGF) has been demonstratedby a previous study (Kamiyama et al., 2003). Inhibition of ROCKs abol-ishes the PDGF-induced activation of extracellular-regulated kinase 1/2(ERK1/2) and proliferation of VSMCs. Moreover, inhibition of ROCKsalso suppresses VSMC proliferation mediated by G-protein-coupledreceptor-stimulated cell proliferation which is promoted by many medi-ators 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 signalingIn endothelium, nitric oxide (NO) is synthesized from the conver-
sion of L-arginine to L-citrulline in the presence of endothelial nitricoxide synthase (eNOS) by using oxygen, NADPH, and substrates(Moncada et al., 1989). After diffusing into the VSMCs, NO activatessoluble guanylate cyclase (sGC), which catalyzes the formation of
guanosine monophosphate (cGMP) and the subsequent activation ofcGMP-dependent protein kinase (cGK). The effect of NO/cGK cascadesignalization in VSMC relaxation results from the decrease of [Ca2+]iintracellular due to the activation of the sarcoplasmatic Ca2+-ATPase,reducing Ca2+ release from intracellular store and the phosphorylationand activation of MLP20. In addition, in VSMCs, the role of eNOS/NO sig-nalization as antiproliferation has been demonstrated (Emerson et al.,1999; Keil et al., 2002).
Recent studies showed that Rho-kinase/ROCKs might negativelyregulate 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 mRNAstability and eNOS expression (Rikitake et al., 2005). ROCK inhibi-tors also enhance phosphorylation and activation of the Akt/phos-phatidylinositol-3 kinase (PI-3K) pathway, leading to increase NOproduction (Wolfrum et al., 2004). In addition, the pleiotropic effectof statins, a nonselective ROCKs inhibitors, on upregulation of eNOShas been demonstrated (Ni et al., 2001; Girgis et al., 2003;Nishimura et al., 2003). Statins upregulate eNOS/NO signalization
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 iscontrolled 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 trans-locates into the membrane and activates its downstream effectors: Rho-kinase or ROCKs. ROCKs increase the vasoconstriction by inhibiting the activity of MLCP via the phosphor-ylation of this enzyme.
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via the inhibition of geranylgeranylation of the small G-protein Rho,and translocate inactive Rho form the cytosol to themembrane (Liao& Laufs, 2005).
4.2.2. Endothelial nitric oxide synthase/nitric oxide signaling and GTPase-RhoA/Rho-kinase
On the other hand, the effect of eNOS/NO signalization on RhoA/Rho-kinase pathway has been recently studied. Previous studiesfounded that NO through cGMP/cGK might serine-phosphorylateRhoA leading to decrease the association of RhoA protein on cellmembrane (Sauzeau et al., 2000; Begum et al., 2002; Chitaley &Webb, 2002; Krepinsky et al., 2003). As a result, it reducedRhoA-GTP active form, and therefore, decreased the activity ofdownstream target effects of ROCKs. Sauzeau et al. (Sauzeau et al.,2000) showed that exogenous NO attenuated Ca2+-dependentROCKs sensitization of blood vessel contraction by inhibiting RhoAtranslocation from the cytosol to membrane in VSMCs via cGK path-way. Moreover, NOmight also inhibit RhoA/ROCKs independently ofcGMP by S-nitrosating RhoA via interaction with cysteine to formnitrothiols (Hess et al., 2001; Stamler et al., 2001; Zuckerbraun etal., 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 principallystudied in chronic hypoxia (CH)-induced PH and monocrotaline(MCT)-induced PH (Table 1). Chronic hypoxia due to different disor-ders or pathologies, is one of the major causes of PH. Exposure tochronic hypoxia induces structural and functional changes in pulmo-nary arterial bed (Rabinovitch et al., 1979; Pierson, 2000) leading toendothelial dysfunction (Higenbottam & Laude, 1998), and modifica-tion of pulmonary vascular contractile properties (Yuan et al., 1998)as described above. Furthermore, in hypoxia-induced PH, the balanceof vasoconstriction–vasodilatation tonus has been impaired by other
vasoconstrictors such as ET-1, 5-HT, Ang II. These vasoconstrictorsalso mediate RhoA/Rho-kinase pathway (Barman, 2007; Homma etal., 2007), and it in turn, is involved in downregulation of eNOS sig-nalization, VSMC hyperconstriction (Batchelor et al., 2001; Sakuradaet al., 2001; Nagaoka et al., 2004, 2006; McNamara et al., 2008;Desbuards et al., 2009), and pulmonary vascular remodeling (Laufset al., 1999a; Sauzeau et al., 2001; Keil et al., 2002). It is known thatdistal muscularization of pulmonary circulation has been increasedduring chronic hypoxia and RhoA/Rho-kinase pathway is involvedin 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 singlesubcutaneous or intraperitoneal of monocrotaline (MCT). Althoughthe exact mechanism though which MCT-induced PH is not wellunderstood. It is suggested that injection of MCT-induced severe orlethal PH is mediated by its direct toxic effect on endothelial cellsand dramatic accumulation of mononuclear inflammatory cells, par-ticularly macrophage, in the small intraacinar vessels (Wilson et al.,1989; Jasmin et al., 2001). The role of Rho-kinase in MCT-inducedPH in rats has been firstly demonstrated by Khan et al. (2005). Inthis study, the authors showed that while pulmonary arterialcontraction induced by noradrenaline was attenuated and due topoor-coupling to G-protein-coupled receptor, the Rho-kinase activ-ity was increased with principally ROCK-2. Increase of Rho-kinaseactivity, 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 asym-metric dimethylarginine (ADMA) which is involved in vascular re-modeling in MCT-induced PH, is mediated by Rho-kinase activity(Li et al., 2010).
Rho-kinase signaling pathway also plays a important role in thepathogenesis of other models of PH. Li F et al. demonstrated that Wis-ter rats with carotid-external jugular vein communication, a model ofhigh-pressure systemic–to-pulmonary shunts, developed PH after 4wk with excessive vascular remodeling at week 8 (Li et al., 2007). In
this study, Rho-kinase signaling pathway was involved in increasedproliferation and decreased apoptosis of SMCs. In severe PHmodel in-duced by hypoxia associated with injected Sugen 5416, a VEGF recep-tor inhibitor, Oka et al. (2007) founded that Rho-kinase activity had acrucial role in the development of occlusive pulmonary vascular le-sions and sustained vasoconstriction. The role of Rho-kinase signalingpathway in bleomycin-induced PH has been demonstrated (Hemneset al., 2008). It is speculated that in this model, Rho-kinase activityis mediated by enhanced reactive oxygen species (ROS), resultinglung fibrosis and NOS uncoupling. Another role of Rho-kinase in ani-mal model with PH has been also reported (Gien et al., 2008).
5.2. In humans
Although the role of Rho-kinase signaling pathway in pathogene-sis of PH has been confirmed in diverse animal models, its role inhuman pulmonary hypertension is not well studied. The first pub-lished study in this field realized by Guilluy et al. (2009), demonstrat-ed that RhoA/Rho-kinase activity was increased in patients withidiopathic pulmonary arterial hypertension (iPAH). The upregulationof RhoA/Rho-kinase in these patients has been mediated by 5-HT viathe serotonylation of Rho protein, contributing to the proliferation ofpulmonary arterial SMCs. In the study from patients with pulmonaryhypertension who underwent lung transplantation, Do et al. (2009)
showed that Rho-kinase activity was correlated to severity (meanpulmonary arterial pressure, mPAP) and evolution of the disease. Inthis study, Rho-kinase activity was involved in pulmonary arterialhypercontraction 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 hypertensionY-27632 [−R-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane-
carboxamide dihydrochloride]] is one of the compounds derived frompyridine with potent ROCK inhibitory effect (Uehata et al., 1997).Y-27632 is a non-specific inhibitor of ROCKs, competing with ATP forbinding to ROCK catalytic sites (Ishizaki et al., 2000). It has also a potentinhibitory effect of Rho-dependent protein kinase C (Uehata et al.,1997).
In the initial study on the effect of inhibition of Rho-kinase in micewith CH-induced PAH, Fagan et al. (2004) reported that Y-27632 de-creased pulmonary vasoconstriction via mediating Ca2+ sensitizationand attenuated the risk of developing PH and vascular remodeling.The study showed that Y-27632 at 30 mg−1.day−1 reduced right ven-tricle systolic pressure (RVSP), right ventricle hypertrophy (RV hy-pertrophy), and neomuscularization of the distal pulmonaryvasculature. Particularly, treatment with Y-27632 increased the ex-pression 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 vascularremodeling 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 ofCH-induced PH, Y-27632 decreased the pulmonary arterial imped-ance, the diameter of main pulmonary arterial, and the right pulmo-nary compliance.
However, Y-27632 is not a high selective pulmonary vasodilator be-cause of its effects on systemic circulation when used intravenously ororally. To avoid the systemic vasodilator effect of Y-27632, Nagaoka etal. (2005) demonstrated that inhalation of aerosolized Y-27632 had aselective 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-27632lasted more than 5 h after 5 min of inhalation. Especially, its effect wasstronger than that with inhalation of NO.
6.1.2. Fasudil in the treatment of pulmonary hypertensionFasudil (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 in-hibitor, competing with ATP for the binding to the kinase (Davies etal., 2000). After oral administration, fasudil is metabolized in hydroxylfasudil (HA-1100), having more selective inhibitory effect on ROCKs(Shimokawa et al., 1999).
Fasudil is more effective than Y-27632 in prevention of CH-inducedPH (Shimokawa, 2002; Shimokawa et al., 2002; Abe et al., 2006). Thefirst use of fasudil in the treatment of PH in animal model has been dem-onstrated byAbe et al. (2004). In this study, the long-term treatmentwithfasudil improved hemodynamic parameters (PAP, RVSP, RV hypertro-phy), pulmonary vascular remodelingwith suppression of VSMCprolifer-ation 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 MCT-induced 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 demonstrat-ed in othermodel of PH (Table 2). InWistar ratswith highflow-inducedPH (Li et al., 2007), treatment with fasudil suppressed Rho-kinase
360 S. Duong-Quy et al. / Pharmacology & Therapeutics 137 (2013) 352–364
hyperactivity, leading to improve PAP, RV hypertrophy, and pulmonaryvascular remodeling. These results were similar to CH-induced PH orsevere occlusive PH, and left-ventricular dysfunction-induced PH(Yamakawa et al., 2000; Guilluy et al., 2005; Abe et al., 2006; Nagaokaet al., 2006; Oka et al., 2007; Ziino et al., 2010; Dai et al., 2011). Particu-larly, fasudil improved the expression of eNOS (Abe et al., 2006) andnormalized pulmonary vascular resistance (PVR) in PH refractory to
Table 2Effects of Rho-kinase inhibitors in PH animal model.
Authors,Year
PH model Rho-kinaseinhibitors(daily dose)
Effect of Rho-kinaseinhibitors
(Vanderpoolet al.,2011)
CH-induced PH inmice
Y-27632 (10−5M, isolatedmouse lungs)
Decreased: PA impedance,diameter of main PA, andright PA compliance
(Dai et al.,2011)
Left-ventriculardysfunction-inducedPH in rats
Fasudil(30 mg/kg)
Improved: mPAP, RVhypertrophy, PA medialthickness
(Xu et al.,2010)
CH-induced PH injuvenile rats
Y-27632(15 mg/kg)
Attenuated: RVhypertrophy, PA wallremodeling; normalized:RVSF
PH, pulmonary hypertension; CH, chronic hypoxia; PA, pulmonary artery; SMCs, smoothmuscle cells; MCT, monocrotaline; RV, right ventricle; RVSF, right ventricular systolicfunction; RVSP, right ventricular systemic pressure; mPAP, mean pulmonary arterialpressure; TPR, total pulmonary resistance; PVR, pulmonary vascular resistance.
nitric oxide (NO) (McNamara et al., 2008). In Fawn-Hooded Rat (FHR)with severe PHwhen raised for the first weeks of life in themild hypox-ia of Denver's altitude (Nagaoka et al., 2006), long-term treatment withfasudil reduced the development of PH and improved lung dysplasia inrat pups by decreasing alveolar size and increasing pulmonary vasculardensity.
Moreover, fasudil might be used in combination with other vaso-dilators in the treatment of PH. Tawara et al. (2007) showed that inMCT-induced PH in rats, when compared with monotherapy, thecombination of fasudil and prostacyclin analogue (beraprost sodium)significantly improved mPAP, RV hypertrophy, and pulmonary arteri-al medial thickness without any adverse effects. This result suggestedthat combined therapy between a Rho-kinase inhibitor with other va-sodilators might exert more beneficial effects in PH treatment due tothe additive and synergic effects of combined treatment.
In addition, fasudil when used by inhalation is as effective as in-haled Y-27632 in the treatment of PH. In CH- and MCT-induced PHin rats, inhaled fasudil reduced significantly mPAP, without any sig-nificant change in systemic arterial pressure and cardiac index orheart rate (Nagaoka et al., 2005). This route of administration is oneof the advantages of fasudil in the treatment of PH and it should beexplored in the future.
6.1.3. Other Rho-kinase inhibitors in the treatment of pulmonary hypertensionAlthough many other new Rho-kinase inhibitors have been devel-
oped, their role in the treatment of PH has not been completely studied.Recently, the therapeutic efficacy of azaindole-1, a potent ROCK inhibi-tor with highly specific ATP-competition, in MCT- and CH-induced PHin rodent has been demonstrated (Dahal et al., 2010). Oral administra-tion of azaindole-1 improved the hemodynamic, RV hypertrophy andpulmonary vascular remodeling. In this study, azaindole-1 had a potenteffect improving a hypoxic pulmonary vasoconstriction and a pulmo-nary arterial SMC proliferation.
Beside the effects of selective Rho-kinase inhibitors in the treat-ment of PH, the role of statin, a non selective Rho-kinase inhibitorwith pleiotropic effect, in the treatment of PH has been demonstrated(Girgis et al., 2007).
6.2. In humans
Successful results of Rho-kinase inhibitors in the treatment of PHin animal model encourage many researchers try to study it inhuman. The first clinical essays of Rho-kinase inhibitors in treatmentof PH have been initiated by Fukumoto et al. (2005) in a small num-ber of enrolled patients (9 patients, mean age of 53 years). The re-sults of this study showed that intravenous administration offasudil (30 mg for 30 minutes) decreased PAP and increased cardiacindex (CI). It also reduced PVR significantly without any side effecton systemic pressure. This result is similar to what reported byIshikura et al. in patients with idiopathic pulmonary arterial hyper-tension (iPAH) and associated PAH (Ishikura et al., 2006).
Recently, the acute beneficial effects of fasudil on hemodynamic in thetreatment of congenital heart diseasewith left-to-right shunt-induced PHin young patients have been demonstrated by a prospective study (12patients, mean age of 12.3 years) (Li et al., 2009). In this study, intrave-nous injection of fasudil (30 mg over 30 min) significantly decreasedPAP and PVR and increased cardiac output, whereas systemic arterialpressure were slightly reduced as compared to baseline value. Fujita etal. showed that the use of fasudil by inhalation in the treatment of PH(iPAH, aPAH, and PAH due to left heart dysfunction) decreased mPAPand the ratio of pulmonary vascular resistance/systemic vascular resis-tance (Fujita et al., 2010). However, the long term effect of fasudil inpatientswith PHasmonotherapy or in combinationwith other treatmenthas not been demonstrated (Table 3).
Table 3Effects of Rho-kinase inhibitors in patients with PAH.
Authors,Year
PH model Rho-kinase inhibitors Effect ofRho-kinaseinhibitors
More than thirty years after the first meeting of PH in Geneva, upto now, PH is still an incurable disease with dramatic survival. De-spite the combination of available vasodilator drugs at early stageof disease, the prognostic of PH remains unfortunately the same assome progressed cancer. The new findings on pathophysiology andpathobiology of PH help physicians improving advanced knowledgeon pathogenesis of this disease. Recent discovery on the role ofRhoA/Rho-kinase signaling pathway in patients with PH and the ef-fects of Rho-kinase inhibitors in vivo promise a novel pharmacolog-ical treatment of PH. These selective Rho-kinase inhibitors areaimed to improve the pulmonary arterial relaxation and remodeling.However, the long-term outcome of clinical trials of Rho-kinase in-hibitors in patients with PHwill be necessary in the future to confirmits 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 interestrelated to this work.
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