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Invited Review

Rho Kinase (ROCK) Inhibitors

Liao, James K MD*; Seto, Minoru PhD; Noma, Kensuke MD, PhD*

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Journal of Cardiovascular Pharmacology: July 2007 - Volume 50 - Issue 1 - p 17-24
doi: 10.1097/FJC.0b013e318070d1bd
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The small GTP-binding proteins belonging to the Rho family regulate various aspects of cell shape, motility, proliferation, and apoptosis.1,2 Rho kinases (ROCKs), which were the first downstream effectors of Rho to be discovered,3-5 were found to mediate RhoA-induced actin cytoskeletal changes through effects on myosin light chain phosphorylation.6,7 ROCKs are protein serine/threonine kinases that share 45% to 50% homology to other actin cytoskeletal kinases such as myotonic dystrophy kinase (DMPK), myotonic dystrophy-related cdc42-binding kinase (MRCK), and citron kinase.1 ROCKs consist of an amino-terminal kinase domain, followed by a mid coiled-coil-forming region containing a Rho-binding domain (RBD), and carboxy-terminal cysteine-rich domain (CRD) located within the pleckstrin homology (PH) motif. Two ROCK isoforms have been identified in mammalian system. ROCK1, which is also known as ROKβ and p160ROCK, is located on chromosome 18 and encodes a 1354-amino acid protein.5,6ROCK2, which is also known as ROKα and sometimes confusingly called Rho-kinase, is located on chromosome 12 and contains 1388 amino acids.3,4,8 ROCK1 and ROCK2 share an overall 65% homology in amino-acid sequence and 92% homology in their kinase domains (Figure 1).

Structures of ROCK isoforms. Both ROCKs consist of an amino-terminal kinase domain followed by a coiled-coil forming region containing a Rho-binding (RB) domain and a carboxy-terminal cystein-rich domain (CRD) located within the plecktrin-homology (PH) domain. ROCK1 and ROCK2 share overall 65% homology in amino acid sequence and 92% homology in their kinase domains.

The carboxy-terminal regions of ROCKs serve as an autoregulatory inhibitor of the amino-terminal kinase domain.9 The interaction of the active GTP-bound form of Rho to ROCK's RBD increases ROCK activity through derepression of the carboxyl-terminal RBD-PH domain on the amino-terminal kinase domain, leading to an active “open” kinase domain. The open conformation could also be caused by the binding of arachidonic acid to the PH domain10 or cleavage of the carboxyl-terminus in ROCK1 by caspase-311,12 and that in ROCK2 by granzyme B or caspase-2.13,14 This closed-to-open conformation of ROCK activation is similar to that of DMPK and MRCK activation9,15 and is consistent with studies showing that overexpression of various carboxyl-terminal constructs of ROCK or kinase-defective forms of full-length ROCK, functions as dominant-negative ROCK mutants.5,6,16 ROCKs can also be activated independently of Rho through amino-terminal transphosphorylation15,17 or inhibited by other small GTP-binding proteins such as Gem and Rad.18

Downstream Targets of ROCK

In response to activators of Rho, such as lysophosphatidic acid (LPA) or sphingosine-1 phosphate (S1P), which stimulate Rho guanine nucleotide exchange factor (GEF) and lead to the formation of active GTP-bound Rho, ROCKs mediate a broad range of cellular responses that involve the actin cytoskeleton. For example, they control assembly of the actin cytoskeleton and cell contractility by phosphorylating a variety of proteins, such as myosin light chain (MLC) phosphatase, LIM kinases, adducin, and ezrin-radixin-moesin (ERM) proteins (Figure 2). These actin cytoskeletal proteins are also phosphorylated by other serine-threonine kinases such as protein kinase A, protein kinase C, and G-kinase.19,20 The consensus amino acid sequences for phosphorylation are R/KXS/T or R/KXXS/T (R: arginine; K: lysine; X: any amino acid; S: serine; T: threonine).21,22 ROCKs can also be auto-phosphorylated,3,5 which might modulate their function.

Regulation of cellular function by ROCK. Stimulation of G-protein-coupled receptors (GPCR) leads to an increase in intracellular calcium/calmodulin (CaM)-mediated activation of myosin light chain kinase (MLCK). MLCK phosphorylates MLC, leading to actin-myosin interaction and cellular contraction, migration, proliferation, and survival. Stimulation of GPCR also leads to ROCK activation via Rho guanine exchange factor (GEF). Activated ROCK, mediated through, phosphorylates various downstream targets, such as ezrin-radixin-moesin (ERM), a 17-kDa PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI17), and the myosin-binding subunit (MBS) of MLC phosphatase. Phosphorylation of MBS inhibits MLC phosphatase activity leading to increase MLC phosphorylation and actomyosin activation. ILK, integrin-linked kinase.

Despite having similar kinase domain, ROCK1 and ROCK2 may serve different functions and may have different downstream targets. Specifically, ROCK2 phosphorylates Ser19 of MLC, the same residue that is phosphorylated by MLC kinase (MLCK). Thus, ROCK2 can alter the sensitivity of SMC contraction to Ca2+ since MLCK is Ca2+-sensitive.23 In addition, ROCKs regulate MLC phosphorylation indirectly through the inhibiton of MLC phosphatase (MLCP) activity. MLCP holoenzyme is composed of 3 subunits: a catalytic subunit (PP1∂), a myosin-binding subunit (MBS) composed of a 58-kD head and 32-kD tail region, and a small non-catalytic subunit, M21. Depending on the species, ROCK2 phosphorylates MBS at Thr697, Ser854, and Thr855.22 Phosphorylation of Thr697 or Thr855 attenuates MLCP activity10 and in some instances, the dissociation of MLCP from myosin.24 ROCK2 also phosphorylates ERM proteins, namely Thr567 of ezrin, Thr564 of radixin, and Thr558 of moesin.25 ROCK-mediated phosphorylation leads to the disruption of the head-to-tail association of ERM proteins and actin cytoskeletal reorganization. In contrast, ROCK1 phosphorylates LIM kinase-1 at Thr508 and LIM kinase-2 at Thr505,21,26 which enhance the ability of LIM kinases to phosphorylate cofilin.27 Since cofilin is an actin-binding and actin-depolymerizing protein that regulates the turnover of actin filaments, the phosphorylation of LIM kinases by ROCKs inhibits cofilin-mediated actin filament disassembly and leads to an increase in the number of actin filaments. Further studies concerning the physiological role of these downstream targets of ROCKs are expected with great respects.

Cellular Functions of ROCK

ROCKs are important regulators of cellular growth, migration, metabolism, and apoptosis through control of the actin cytoskeletal assembly and cell contraction.1 Although there is no evidence that ROCK isoforms have different functions, they are differentially expressed and regulated in various tissues. For example, only ROCK1 is cleaved by caspase-3 during apoptosis,11,12 while smooth muscle-specific basic calponin is phosphorylated only by ROCK2.28 Furthermore, ROCK1 expression tends to be more ubiquitous, while ROCK2 is most highly expressed in cardiac and brain tissues.8,29,30 Indeed, homozygous deletion of ROCK1 and ROCK2 leads to differing causes of embryonic lethality.31,32 Thus, it is likely that using a genetic approach to dissecting the roles of ROCK isoforms (ie, conditional ROCK deletion), distinct and novel cellular functions will be uncovered, which could be specifically ascribed to either ROCK1 or ROCK2.

There is growing evidence that abnormal ROCK function contributes to cardiovascular disease.

Stimulation of tyrosine kinase and G-protein-coupled receptors leads to activation of Rho, the direct upstream activator of ROCKs, via recruitment and activation of RhoGEF.33,34 ROCKs are important effectors of Rho in regulating the actin cytoskeleton. Inhibitors of ROCKs, such as Y27632 and fasudil, or overexpression of dominant-negative mutants of ROCKs lead to the loss of stress fibers and focal adhesion complexes.5,35 This is due predominantly to the phosphorylation and inhibition of MLCP by ROCK, which increases MLC phosphorylation and cellular contraction, by facilitating interaction of myosin with F-actin (Figure 2). Thus, ROCKs regulate cell polarity and migration predominantly through enhancing actomyosin contraction and focal adhesions. This would increase cellular contraction as well as mediate cellular migration and chemotaxis. Indeed, increased ROCK activity is observed in tumor metastasis36 and overexpression of constitutively activated ROCK promotes tumor invasion.37 ROCK also regulates leukocyte chemotaxis, possibly by altering the localization and activation of phosphatase and tensin homologue (PTEN).38,39 Conversely, invasion of rat hepatoma cells and migration of metastatic breast cancer cells are inhibited by overexpression of dominant-negative ROCK constructs or by the ROCK inhibitor Y-27632.40 Treatment with Y-27632 reduces tumor-cell dissemination in vivo, suggesting its potential use in cancer therapy.40 In addition, ROCKs could also regulate macrophage phagocytic activity via actin cytoskeletal membrane protrusions and mediate endothelial cell permeability via affects on tight and adheren junctions.41,42 ROCKs could inhibit insulin signaling via phosphorylation of insulin receptor substrate (IRS)-1, which uncouples the insulin receptor to phosphatidylinositol-3 kinase.43 Conversely, it could also regulate cell size via enhancing insulin-like growth factor (IGF)-induced cAMP response element binding protein (CREB) phosphorylation.44 Indeed, this may be the underlying mechanism by which ROCK inhibitors reduce cardiac hypertrophy.45,46 Finally, ROCKs may be involved in tissue differentiation from adipocytes to myocytes. In p190-B Rho GTPase-activating protein (GAP)-deficient mice, which have high basal Rho/ROCK activity because there is no “off switch” for Rho, there is a defect in adipogenesis, with a predilection toward myogenesis.44,47 Treatment of p190-B RhoGAP-deficient mice with Y27632 restores normal adipogenesis,47 suggesting that ROCKs are involved in the myogenesis differentiation program.

Role of ROCK in Cardiovascular Disease

Many cholesterol-independent or so-called “pleiotropic” effects 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors or statins are due to their ability to block the synthesis of isoprenoid intermediates, which serve as important lipid attachments for a variety of intracellular signaling molecules.48 In particular, the inhibition of small GTP-binding proteins Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation,48 may play an important role in mediating the biological effects of statins. For example, statins increase the expression of endothelial nitric oxide synthase (eNOS) via inhibition of RhoA/ROCK-mediated actin cytoskeletal changes, leading to the stabilization of eNOS mRNA.49,50 Indeed, a recent report suggests that binding of G-actin to the 3′-untranslated region of eNOS mRNA decreases eNOS mRNA expression.51 Futhermore, inhibition of Rho/ROCK pathway leads to the rapid phosphorylation and activation of eNOS via the phosphatidylinositol (PI)-3 kinase/protein kinase Akt pathway.52,53 Thus, Rho/ROCKs negatively regulate endothelial function at the level of both eNOS expression and activation via 2 distinct mechanisms.

ROCK activity is involved in the expression of PAI-1 mediated by hyperglycemia, indicating that ROCK may function as a key regulator of cardiovascular injury in patients with diabetes mellitus.

There is growing evidence that abnormal ROCK function contributes to cardiovascular disease.54 In the vascular wall, ROCK mediates vascular smooth muscle contraction, actin cytoskeleton organization, cell adhesion, and motility.55 Thus, abnormal ROCK activity may contribute to abnormal smooth muscle contraction observed in cerebral and coronary vasospasm,56,57 hypertension,58 and pulmonary hypertension.59 In addition, ROCK could also regulate vascular tone and blood flow indirectly through negative effects on eNOS expression and activity52,60 or via direct effects on the central nervous system.61,62 Inhibition of ROCK leads to increase in cerebral blood flow and decrease in cerebral infarct size via upregulation of eNOS.63 ROCK is also involved in vascular inflammation and remodeling,64 restenosis after balloon injury,65-67 ischemia-reperfusion injury,52,68,69 and atherosclerosis.70,71 Recent studies also suggest that long-term treatment with a ROCK inhibitor, fasudil, improved monocrotaline-induced fatal pulmonary hypertension in rats59 and suppresses cardiac allograft vasculopathy in mice.72 ROCK has also been implicated in the expression of a variety of genes, which are pertinent to vascular function, such as monocyte chemoattractant protein-1 (MCP-1),73 plasminogen activator inhibitor-1 (PAI-1),74 and osteopontin.75 Indeed, ROCK is upregulated by inflammatory stimuli, such as angiotensin II and interleukin-1β, in cultured cells76 and by lipopolysaccharide (LPS) in vivo.77

Rationale for the Development of ROCK Inhibitors

Despite an increasing number of reports showing that ROCK activity is increased under a variety of pathological conditions, little is known about the molecular mechanisms that contribute to increased ROCK activity or what the downstream targets for ROCK are. Furthermore, determining the precise role of ROCK in the vascular wall is limited by pharmacological inhibitors, which cannot discriminate between ROCK isoforms or the role of ROCKs in individual component cells. Hence, a genetic approach with tissue-specific gene targeting of specific ROCK deletion to individual components of the vascular wall offers the greatest likelihood of success in dissecting the role of pathophysiological role of ROCKs. Because ROCKs are critical for cardiovascular and central nervous system (CNS) development, embryonic lethality occurs in both ROCK1−/− and ROCK2−/− mice.31,78 However, the phenotypes of the lethality are quite different. ROCK1−/− mice die soon after birth due to development of omphalocoele caused by a defect in umbilical ring closure from impairment of filamentous actin accumulation. ROCK1−/− mice also exhibit eyes open at birth (EOB) due to disorganization of actin filaments in the epithelial cells of the eyelid. In contrast, ROCK2−/− mice die embryonically due to dysfunction and intrauterine growth retardation caused by the manifest thrombus formation in the labyrinth layer of the placenta. These findings suggest distinct tissue distribution and downstream targets of ROCK1 and ROCK2. Indeed, although previous studies suggest that ROCK inhibitors prevent the development of cardiac hypertrophy,78-80 ROCK1−/− mice develop cardiac hypertrophy, but not fibrosis.78,81 It is possible that ROCK2 but not ROCK1 is involved in the development of cardiac hypertrophy. Furthermore, nonselective ROCK inhibitors have been shown to decrease systemic blood pressure.58,82 However, neither the haploinsufficient ROCK1 nor ROCK2 mouse, show any differences in basal or angiotensin II-induced increase in systemic blood pressure compared with that of wild-type mice.78

Despite the potential clinical importance of ROCK inhibition, fasudil is the only ROCK inhibitor approved for human use.

Nevertheless, because ROCK is involved in various aspects of vascular function and inflammatory conditions, the development of selective and nonselective ROCK inhibitors has gained considerable interest in the pharmaceutical industry. Presently, Y-27632 and fasudil are non-isoform-selective ROCK inhibitors that target their ATP-dependent kinase domains and are therefore equipotent in terms of inhibiting both ROCK1 and ROCK2. Neither fasudil nor Y27632 can distinguish between ROCK1 and ROCK2. Furthermore, at higher concentrations, these ROCK inhibitors could also inhibit other serine-threonine kinases such as PKA and PKC.63 Nevertheless, compared with the other kinases, fasudil and its active metabolite, hydroxyfasudil, are relatively more selective for ROCKs, with hydroxyfasudil being slightly more selective than fasudil and Y27632.63 Compared to ROCKs, the IC50 value for PKA was approximately 5-fold higher for fasudil and 50-fold higher for hydroxyfasudil. With the exception of PKC isoforms, which have IC50 values ranging from 20 to 100 μmol/L, all of the other protein kinases tested such as Raf1, ERK, and p38, have IC50 values that were >100 μmol/L for fasudil and hydroxyfasudil.63

ROCK Inhibitors in Cardiovascular Disease

Non-isoform-selective ROCK inhibitors such as fasudil have been shown to prevent cerebral vasospasm after subarachnoid hemorrhage.56,83 Similarly, animal studies with Y-27632 showed that it could inhibit the development of atherosclerosis and arterial remodeling following vascular injury.65,70 ROCK activity is involved in the expression of PAI-1 mediated by hyperglycemia, indicating that ROCK may function as a key regulator of cardiovascular injury in patients with diabetes mellitus.84 Furthermore, RhoA/ROCK pathway has been reported to be involved in angiogenesis,85,86 cerebral ischemia,87 erectile dysfunction,88,89 glomerulosclerosis,90 hypertension,35 myocardial hypertrophy,79 myocardial ischemia-reperfusion injury,52,69 neointima formation,65,91 pulmonary hypertension,59 and vascular remodeling.71 Moreover, ROCK inhibitors have shown benefits in Alzheimer's disease,92 bronchial asthma,93 cancers,94 demyelinating diseases,92 glaucoma,95 and osteoporosis.96 Although most of the previous studies have shown that inhibition of both isoforms by ROCK inhibitors results in the beneficial effect, whether the effects are mediated by inhibition of ROCK1, ROCK2, or both, remains to be determined.

Development of ROCK Inhibitors in Cardiovascular Disease

Inhibition of ROCK by fasudil leads to beneficial effects in patients with systemic hypertension,58 pulmonary hypertension,97 vasospastic angina,57 stable effort angina,98 stroke,99 and chronic heart failure.100 Indeed, perhaps many of the so-called “pleiotropic” effects of statins may be mediated by ROCK inhibition,49,50,52,60,101,102 while the extent of inhibitory effect of ROCK by statins remains to be cleared, especially in humans. However, despite the potential clinical importance of ROCK inhibition, fasudil is the only ROCK inhibitor approved for human use.

The development of fasudil began with functional research on calmodulin inhibition by quinoline or isoquinoline derivatives. Fasudil was obtained by chemical screening of isoquinoline sulfonamide derivatives using a bioassay for vasodilatory activity of normal and spastic arteries. The isoquinoline derivatives are metabolized to their hydroxyl forms in animals and humans. The hydroxyl form of fasudil, hydroxyfasudil, has the same ROCK inhibitory activity as fasudil. Although the half-life of fasudil in humans is extremely short (eg, less than 0.5 hours), the half-life of hydroxyfasudil, which is >5 hours, is of sufficient duration for the efficacy of ROCK inhibition to be observed with twice-daily fasudil administration. Furthermore, the ratios of the protein-unbound form of fasudil and hydroxyfasudil are greater than 50% in human plasma. The large amount of protein-unbound form allows the drug to easily distribute to target organs and may contribute to its efficacy. In 1995, fasudil was approved in Japan and China for prevention and treatment of cerebral vasospasm following surgery for subarachnoid hemorrhage and has since been used in over 124,000 patients in Japan. Although several adverse effects such as hepatic function abnormal, intracranial hemorrhage, and hypotension have been reported, previous investigators have also revealed that no serious adverse events have been seen not only in fasudil-treated patients of subarachnoid hemorrhage but also of acute ischemic stroke compared with those in placebo-treated patients.99,103,104 Hence, as a selective ROCK inhibitor, fasudil is currently being developed for treating acute stroke and pulmonary artery hypertension.

Given the breadth of preclinical data suggesting benefits of ROCK inhibition in hypertension and cardiovascular diseases, many biotechnology and pharmaceutical companies are developing other selective and nonselective ROCK inhibitors (Table 1). Many of these compounds are still in the discovery phase. So, it will be some time before we know whether ROCK isoforms are viable targets in humans at risk for cardiovascular disease.

ROCK Inhibitors


There is growing evidence that RhoA/ROCK pathway plays an important pathophysiological role in cardiovascular diseases and that inhibition of ROCKs by ROCK inhibitors or statins may be beneficial. To date, a great number of cellular and physiological functions are mediated by ROCK, and ROCK activity is often elevated in disorders of the cardiovascular system. Thus, inhibition of ROCK may be an attractive therapeutic target in reducing cardiovascular disease. However, a greater understanding of the physiological role of each ROCK isoforms in the cardiovascular system and the development of isoform-specific inhibitors are needed to resolve the specificity and safety of ROCK inhibitors.


1. Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003;4:446-456.
2. Rikitake Y, Liao JK. Rho GTPases, statins, and nitric oxide. Circ Res. 2005;97:1232-1235.
3. Leung T, Manser E, Tan L, et al. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem. 1995;270:29051-29054.
4. Matsui T, Amano M, Yamamoto T, et al. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. Embo J. 1996;15:2208-2216.
5. Ishizaki T, Maekawa M, Fujisawa K, et al. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. Embo J. 1996;15:1885-1893.
6. Leung T, Chen XQ, Manser E, et al. 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. 1996;16:5313-5327.
7. Somlyo AV, Bradshaw D, Ramos S, et al. Rho-kinase inhibitor retards migration and in vivo dissemination of human prostate cancer cells. Biochem Biophys Res Commun. 2000;269:652-659.
8. Nakagawa O, Fujisawa K, Ishizaki T, et al. ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice. FEBS Lett. 1996;392:189-193.
9. Amano M, Chihara K, Nakamura N, et al. The COOH terminus of Rho-kinase negatively regulates rho-kinase activity. J Biol Chem. 1999;274:32418-32424.
10. Feng J, Ito M, Ichikawa K, et al. Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J Biol Chem. 1999;274:37385-37390.
11. Sebbagh M, Renvoize C, Hamelin J, et al. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol. 2001;3:346-352.
12. Coleman ML, Sahai EA, Yeo M, et al. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol. 2001;3:339-345.
13. Alberts AS, Qin H, Carr HS, et al. PAK1 negatively regulates the activity of the Rho exchange factor NET1. J Biol Chem. 2005;280:12152-12161.
14. Sapet C, Simoncini S, Loriod B, et al. Thrombin-induced endothelial microparticle generation: identification of a novel pathway involving ROCK-II activation by caspase-2. Blood. 2006;108:1868-1876.
15. Chen XQ, Tan I, Ng CH, et al. Characterization of RhoA-binding kinase ROKalpha implication of the pleckstrin homology domain in ROKalpha function using region-specific antibodies. J Biol Chem. 2002;277:12680-12688.
16. Amano M, Chihara K, Kimura K, et al. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science. 1997;275:1308-1311.
17. Turner MS, Fen Fen L, Trauger JW, et al. Characterization and purification of truncated human Rho-kinase II expressed in Sf-21 cells. Arch Biochem Biophys. 2002;405:13-20.
18. Ward Y, Yap SF, Ravichandran V, et al. The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway. J Cell Biol. 2002;157:291-302.
19. Hartshorne DJ. Myosin phosphatase: subunits and interactions. Acta Physiol Scand. 1998;164:483-493.
20. Rikitake Y, Liao JK. ROCKs as therapeutic targets in cardiovascular diseases. Expert Rev Cardiovasc Ther. 2005;3:441-451.
21. Sumi T, Matsumoto K, Nakamura T. Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase. J Biol Chem. 2001;276:670-676.
22. Kawano Y, Fukata Y, Oshiro N, et al. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J Cell Biol. 1999;147:1023-1038.
23. Amano M, Ito M, Kimura K, et al. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem. 1996;271:20246-20249.
24. Velasco G, Armstrong C, Morrice N, et al. Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin. FEBS Lett. 2002;527:101-104.
25. Matsui T, Maeda M, Doi Y, et al. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol. 1998;140:647-657.
26. Ohashi K, Nagata K, Maekawa M, et al. Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. J Biol Chem. 2000;275:3577-3582.
27. Maekawa M, Ishizaki T, Boku S, et al. Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science. 1999;285:895-898.
28. Kaneko T, Amano M, Maeda A, et al. Identification of calponin as a novel substrate of Rho-kinase. Biochem Biophys Res Commun. 2000;273:110-116.
29. Di Cunto F, Imarisio S, Hirsch E, et al. Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis. Neuron. 2000;28:115-127.
30. Wei L, Roberts W, Wang L, et al. Rho kinases play an obligatory role in vertebrate embryonic organogenesis. Development. 2001;128:2953-2962.
31. Thumkeo D, Keel J, Ishizaki T, et al. Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death. Mol Cell Biol. 2003;23:5043-5055.
32. Shimizu Y, Thumkeo D, Keel J, et al. ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles. J Cell Biol. 2005;168:941-953.
33. Hart MJ, Jiang X, Kozasa T, et al. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. Science. 1998;280:2112-2114.
34. Kozasa T, Jiang X, Hart MJ, et al. p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13. Science. 1998;280:2109-2111.
35. Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997;389:990-994.
36. Bourguignon LY, Zhu H, Shao L, et al. Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells. Cell Motil Cytoskeleton. 1999;43:269-287.
37. Yoshioka K, Nakamori S, Itoh K. Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells. Cancer Res. 1999;59:2004-2010.
38. Li Z, Dong X, Wang Z, et al. Regulation of PTEN by Rho small GTPases. Nat Cell Biol. 2005;7:399-404.
39. Thorlacius K, Slotta JE, Laschke MW, et al. Protective effect of fasudil, a Rho-kinase inhibitor, on chemokine expression, leukocyte recruitment, and hepatocellular apoptosis in septic liver injury. J Leukoc Biol. 2006;79:923-931.
40. Itoh K, Yoshioka K, Akedo H, et al. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat Med. 1999;5:221-225.
41. Wojciak-Stothard B, Ridley AJ. Rho GTPases and the regulation of endothelial permeability. Vascul Pharmacol. 2002;39:187-199.
42. Wojciak-Stothard B, Potempa S, Eichholtz T, et al. Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci. 2001;114:1343-1355.
43. Farah S, Agazie Y, Ohan N, et al. A rho-associated protein kinase, ROKalpha, binds insulin receptor substrate-1 and modulates insulin signaling. J Biol Chem. 1998;273:4740-4746.
44. Sordella R, Classon M, Hu KQ, et al. Modulation of CREB activity by the Rho GTPase regulates cell and organism size during mouse embryonic development. Dev Cell. 2002;2:553-565.
45. O'Cochlain DF, Perez-Terzic C, Reyes S, et al. Transgenic overexpression of human DMPK accumulates into hypertrophic cardiomyopathy, myotonic myopathy and hypotension traits of myotonic dystrophy. Hum Mol Genet. 2004;13:2505-2518.
46. Pan J, Singh US, Takahashi T, et al. PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes. J Cell Physiol. 2005;202:536-553.
47. Sordella R, Jiang W, Chen GC, et al. Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis. Cell. 2003;113:147-158.
48. Liao JK. Isoprenoids as mediators of the biological effects of statins. J Clin Invest. 2002;110:285-288.
49. Laufs U, La Fata V, Plutzky J, et al. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation. 1998;97:1129-1135.
50. Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998;273:24266-24271.
51. Searles CD, Ide L, Davis ME, et al. Actin cytoskeleton organization and posttranscriptional regulation of endothelial nitric oxide synthase during cell growth. Circ Res. 2004;95:488-495.
52. Wolfrum S, Dendorfer A, Rikitake Y, et al. Inhibition of Rho-kinase leads to rapid activation of phosphatidylinositol 3-kinase/protein kinase Akt and cardiovascular protection. Arterioscler Thromb Vasc Biol. 2004;24:1842-1847.
53. Wolfrum S, Grimm M, Heidbreder M, et al. Acute reduction of myocardial infarct size by a hydroxymethyl glutaryl coenzyme a reductase inhibitor is mediated by endothelial nitric oxide synthase. J Cardiovasc Pharmacol. 2003;41:474-480.
54. Shimokawa H. Rho-kinase as a novel therapeutic target in treatment of cardiovascular diseases. J Cardiovasc Pharmacol. 2002;39:319-327.
55. Fukata Y, Amano M, Kaibuchi K. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci. 2001;22:32-39.
56. Sato M, Tani E, Fujikawa H, et al. Involvement of Rho-kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm. Circ Res. 2000;87:195-200.
57. Masumoto A, Mohri M, Shimokawa H, et al. Suppression of coronary artery spasm by the Rho-kinase inhibitor fasudil in patients with vasospastic angina. Circulation. 2002;105:1545-1547.
58. Masumoto A, Hirooka Y, Shimokawa H, et al. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension. 2001;38:1307-1310.
59. Abe K, Shimokawa H, Morikawa K, et al. Long-term treatment with a Rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hypertension in rats. Circ Res. 2004;94:385-393.
60. Takemoto M, Sun J, Hiroki J, et al. Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation. 2002;106:57-62.
61. Ito K, Hirooka Y, Kishi T, et al. Rho/Rho-kinase pathway in the brainstem contributes to hypertension caused by chronic nitric oxide synthase inhibition. Hypertension. 2004;43:156-162.
62. Ito K, Shimomura E, Iwanaga T, et al. Essential role of rho kinase in the Ca2+ sensitization of prostaglandin F(2alpha)-induced contraction of rabbit aortae. J Physiol. 2003;546:823-836.
63. Rikitake Y, Kim HH, Huang Z, et al. Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke. 2005;36:2251-2257.
64. Kataoka C, Egashira K, Inoue S, et al. Important role of Rho-kinase in the pathogenesis of cardiovascular inflammation and remodeling induced by long-term blockade of nitric oxide synthesis in rats. Hypertension. 2002;39:245-250.
65. Sawada N, Itoh H, Ueyama K, et al. Inhibition of rho-associated kinase results in suppression of neointimal formation of balloon-injured arteries. Circulation. 2000;101:2030-2033.
66. Shibata R, Kai H, Seki Y, et al. Rho-kinase inhibition reduces neointima formation after vascular injury by enhancing Bax expression and apoptosis. J Cardiovasc Pharmacol. 2003;42(Suppl 1):S43-47.
67. Eto Y, Shimokawa H, Hiroki J, et al. Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs. Am J Physiol Heart Circ Physiol. 2000;278:H1744-1750.
68. Ikeda F, Terajima H, Shimahara Y, et al. Reduction of hepatic ischemia/reperfusion-induced injury by a specific ROCK/Rho kinase inhibitor Y-27632. J Surg Res. 2003;109:155-160.
69. Bao W, Hu E, Tao L, et al. Inhibition of Rho-kinase protects the heart against ischemia/reperfusion injury. Cardiovasc Res. 2004;61:548-558.
70. Mallat Z, Gojova A, Sauzeau V, et al. Rho-associated protein kinase contributes to early atherosclerotic lesion formation in mice. Circ Res. 2003;93:884-888.
71. Miyata K, Shimokawa H, Kandabashi T, et al. Rho-kinase is involved in macrophage-mediated formation of coronary vascular lesions in pigs in vivo. Arterioscler Thromb Vasc Biol. 2000;20:2351-2358.
72. Hattori T, Shimokawa H, Higashi M, et al. Long-term treatment with a specific Rho-kinase inhibitor suppresses cardiac allograft vasculopathy in mice. Circ Res. 2004;94:46-52.
73. Funakoshi Y, Ichiki T, Shimokawa H, et al. Rho-kinase mediates angiotensin II-induced monocyte chemoattractant protein-1 expression in rat vascular smooth muscle cells. Hypertension. 2001;38:100-104.
74. Takeda K, Ichiki T, Tokunou T, et al. Critical role of Rho-kinase and MEK/ERK pathways for angiotensin II-induced plasminogen activator inhibitor type-1 gene expression. Arterioscler Thromb Vasc Biol. 2001;21:868-873.
75. Kawamura H, Yokote K, Asaumi S, et al. High glucose-induced upregulation of osteopontin is mediated via Rho/Rho kinase pathway in cultured rat aortic smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004;24:276-281.
76. Hiroki J, Shimokawa H, Higashi M, et al. Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells. J Mol Cell Cardiol. 2004;37:537-546.
77. Buyukafsar K, Arikan O, Ark M, et al. Upregulation of Rho-kinase (ROCK-2) expression and enhanced contraction to endothelin-1 in the mesenteric artery from lipopolysaccharide-treated rats. Eur J Pharmacol. 2004;498:211-217.
78. Rikitake Y, Oyama N, Wang CY, et al. Decreased perivascular fibrosis but not cardiac hypertrophy in ROCK1+/− haploinsufficient mice. Circulation. 2005;112:2959-2965.
79. Higashi M, Shimokawa H, Hattori T, et al. Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo: effect on endothelial NAD(P)H oxidase system. Circ Res. 2003;93:767-775.
80. Masamune A, Kikuta K, Satoh M, et al. Rho kinase inhibitors block activation of pancreatic stellate cells. Br J Pharmacol. 2003;140:1292-1302.
81. Zhang YM, Bo J, Taffet GE, et al. Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis. Faseb J. 2006;20:916-925.
82. Mukai Y, Shimokawa H, Matoba T, et al. Involvement of Rho-kinase in hypertensive vascular disease: a novel therapeutic target in hypertension. Faseb J. 2001;15:1062-1064.
83. Katsumata N, Shimokawa H, Seto M, et al. Enhanced myosin light chain phosphorylations as a central mechanism for coronary artery spasm in a swine model with interleukin-1beta. Circulation. 1997;96:4357-4363.
84. Rikitake Y, Liao JK. Rho-kinase mediates hyperglycemia-induced plasminogen activator inhibitor-1 expression in vascular endothelial cells. Circulation. 2005;111:3261-3268.
85. Kishore R, Qin G, Luedemann C, et al. The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-alpha-induced transcriptional repression of cyclin A. J Clin Invest. 2005;115:1785-1796.
86. Hyvelin JM, Howell K, Nichol A, et al. Inhibition of Rho-kinase attenuates hypoxia-induced angiogenesis in the pulmonary circulation. Circ Res. 2005;97:185-191.
87. Toshima Y, Satoh S, Ikegaki I, et al. A new model of cerebral microthrombosis in rats and the neuroprotective effect of a Rho-kinase inhibitor. Stroke. 2000;31:2245-2250.
88. Chitaley K, Wingard CJ, Clinton Webb R, et al. Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway. Nat Med. 2001;7:119-122.
89. Bivalacqua TJ, Champion HC, Usta MF, et al. RhoA/Rho-kinase suppresses endothelial nitric oxide synthase in the penis: a mechanism for diabetes-associated erectile dysfunction. Proc Natl Acad Sci USA. 2004;101:9121-9126.
90. Nishikimi T, Akimoto K, Wang X, et al. Fasudil, a Rho-kinase inhibitor, attenuates glomerulosclerosis in Dahl salt-sensitive rats. J Hypertens. 2004;22:1787-1796.
91. Matsumoto Y, Uwatoku T, Oi K, et al. Long-term inhibition of Rho-kinase suppresses neointimal formation after stent implantation in porcine coronary arteries: involvement of multiple mechanisms. Arterioscler Thromb Vasc Biol. 2004;24:181-186.
92. Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for neurological disorders. Nat Rev Drug Discov. 2005;4:387-398.
93. Chiba Y, Misawa M. The role of RhoA-mediated Ca2+ sensitization of bronchial smooth muscle contraction in airway hyperresponsiveness. J Smooth Muscle Res. 2004;40:155-167.
94. Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev. 2003;83:1325-1358.
95. Honjo M, Tanihara H, Inatani M, et al. Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility. Invest Ophthalmol Vis Sci. 2001;42:137-144.
96. Ohnaka K, Shimoda S, Nawata H, et al. Pitavastatin enhanced BMP-2 and osteocalcin expression by inhibition of Rho-associated kinase in human osteoblasts. Biochem Biophys Res Commun. 2001;287:337-342.
97. Fukumoto Y, Matoba T, Ito A, et al. Acute vasodilator effects of a Rho-kinase inhibitor, fasudil, in patients with severe pulmonary hypertension. Heart. 2005;91:391-392.
98. Shimokawa H, Hiramori K, Iinuma H, et al. Anti-anginal effect of fasudil, a Rho-kinase inhibitor, in patients with stable effort angina: a multicenter study. J Cardiovasc Pharmacol. 2002;40:751-761.
99. Shibuya M, Hirai S, Seto M, et al. Effects of fasudil in acute ischemic stroke: results of a prospective placebo-controlled double-blind trial. J Neurol Sci. 2005;238:31-39.
100. Kishi T, Hirooka Y, Masumoto A, et al. Rho-kinase inhibitor improves increased vascular resistance and impaired vasodilation of the forearm in patients with heart failure. Circulation. 2005;111:2741-2747.
101. Laufs U, Fata VL, Liao JK. Inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase blocks hypoxia-mediated down-regulation of endothelial nitric oxide synthase. J Biol Chem. 1997;272:31725-31729.
102. Laufs U, Endres M, Stagliano N, et al. Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J Clin Invest. 2000;106:15-24.
103. Shibuya M, Suzuki Y, Sugita K, et al. Dose escalation trial of a novel calcium antagonist, AT877, in patients with aneurysmal subarachnoid haemorrhage. Acta Neurochir (Wien). 1990;107:11-15.
104. Shibuya M, Suzuki Y, Sugita K, et al. Effect of AT877 on cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Results of a prospective placebo-controlled double-blind trial. J Neurosurg. 1992;76:571-577.

actin cytoskeleton; Rho GTPase; hypertension; inflammation; atherosclerosis

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