Rho-kinase as a therapeutic target in vascular diseases: Striking nitric oxide signaling

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Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x

Rho-kinase as a therapeutic target in vascular diseases: Striking nitric oxide signaling Gopi Krishna Kolluru a, Syamantak Majumder b, Suvro Chatterjee c,d * a

Department of Pathology, LSU Health-Shreveport, USA Aab Cardiovascular Research Institute, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA Department of Biotechnology, Anna University, Chennai, India d Vascular Biology Lab, AU-KBC Research Centre, Anna University, Chennai, India b c

A R T I C L E

I N F O

Article history: Received 23 April 2014 Revised 3 September 2014 Available online Keywords: Rho-GTPases Nitric oxide Shear stress ROCK inhibitors

A B S T R A C T

Rho GTPases are a globular, monomeric group of small signaling G-protein molecules. Rho-associated protein kinase/Rho-kinase (ROCK) is a downstream effector protein of the Rho GTPase. Rho-kinases are the potential therapeutic targets in the treatment of cardiovascular diseases. Here, we have primarily discussed the intriguing roles of ROCK in cardiovascular health in relation to nitric oxide signaling. Further, we highlighted the biphasic effects of Y-27632, a ROCK inhibitor under shear stress, which acts as an agonist of nitric oxide production in endothelial cells. The biphasic effects of this inhibitor raised the question of safety of the drug usage in treating cardiovascular diseases. © 2014 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

7.

Introduction ............................................................................................................................................................................................................................................................. NO and Rho-kinase influence on vascular remodeling ............................................................................................................................................................................. Rho GTPases in cardiovascular health ............................................................................................................................................................................................................ CVD drugs targeting Rho GTPases .................................................................................................................................................................................................................... Shear stress activates eNOS: a defining factor in cardiovascular functions ...................................................................................................................................... ROCK and ROCK inhibitors perturb nitric oxide signaling: an experimental evidence ................................................................................................................. 6.1. Application of shear stress to EC ........................................................................................................................................................................................................ 6.2. Estimation of NO by DAF-FM and NO electrode ........................................................................................................................................................................... 6.3. Real time NO detection using DAF-2DA ........................................................................................................................................................................................... 6.4. Measurements of H2O2 ........................................................................................................................................................................................................................... 6.5. Measurement of peroxynitrite ............................................................................................................................................................................................................ Level of shear stress defines the effects of ROCKI in the endothelium: a clinically relevant issue ........................................................................................... Acknowledgments ................................................................................................................................................................................................................................................. References ................................................................................................................................................................................................................................................................

1. Introduction Rho GTPases are small signaling G-protein molecules with a mass in the range of 20–30 kDa [1]. They have a common G-domain fold, which has a six-stranded β-sheet surrounded by α-helices [2]. They are classified as subfamily under the Ras superfamily. Rho GTPases

* Corresponding author. Vascular Biology Lab, AU-KBC Research Centre, MIT Campus, Anna University, Chennai 600 044, Tamil Nadu, India. Fax: +91 44 2223 1034. E-mail address: [email protected] (S. Chatterjee).

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are present in all eukaryotic organisms including yeasts and some plants [3]. The members of the Rho GTPase family regulate a broad array of activities in the cell such as intracellular actin dynamics, cell polarity, cell cycle progression and cell migration [4]. Thus in the presence of external stimuli Rho GTPases mediate changes in cell morphology and cell motility through the regulation of actin cytoskeleton [5]. Rho-GTPase and its downstream effector, Rhokinase (ROCK), play a central role in angiogenesis by modulating diverse cellular functions such as cytoskeletal rearrangement, cell migration, proliferation and gene expression in endothelial cells (ECs) [6–8]. Rho GTPase also regulates the activity of enzymes involved

http://dx.doi.org/10.1016/j.niox.2014.09.002 1089-8603/© 2014 Elsevier Inc. All rights reserved.

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in lipid metabolism. The effector proteins in lipid metabolism are PI4P 5-kinase [9], PI-3-kinase [10], and DAG kinase [11] etc. Takeya and Sumimoto reported an association between NADPH oxidases and Rho GTPase mediated ROS generation [12]. In mammals, there are approximately 22 known Rho protein members that are divided into subgroups such as Rho, Rac, Cdc42, Rnd, RhoD, RhoF, RhoH, and RhoBTB [13]. Rho subgroup has three members RhoA, RhoB and RhoC that show more than 85% identity in amino acid sequence with basic differences in their C-terminal hypervariable region [14]. Other subgroups have been described as three Rac isoforms 1, 2, and 3; Cdc42, RhoD, Rnd1, Rnd2, RhoE/Rnd3, RhoG, TC10, and TCL; RhoH/TTF; Chp and Wrch-1; Rif, RhoBTB1, and 2; and Miro-1 and 2 [15]. Rho GTPases act as intracellular molecular switches and their activity is regulated by GTP-GDP transformation. When GDP binds with GTPase, the enzyme becomes inactive. GTPase is activated by GEF (guanine nucleotide exchange factor), which is present upstream from the site. GEF causes release of GDP from the enzyme and thus GDP gets replaced by GTP thereby activating the GTPase [16]. It remains active until the hydrolysis of GTP occurs by GAP (GTPase activating protein) enzyme. Rho GTPases also play an important role in the formation of adherent junctions [17]. Allen et al. showed that Cdc42 (subgroup of Rho GTPase) is essential for the directional migration of macrophage in a gradient of chemoattractant [18]. Random migration was observed under inhibited GTPase activity. ROCK, a downstream effector of Rho GTPase (RhoA) is being considered as potential therapeutic target in diseases such as glaucoma, pulmonary hypertension, nerve injury, and cardiac hypertrophy [19] including arterial hypertension, diabetes, erectile dysfunction and vasospasm [20]. Recently, endothelial Rho GTPases are reported as important players in transendothelial migration of blood cells by causing cytoskeleton remodeling [21]. 2. NO and Rho-kinase influence on vascular remodeling Nitric oxide (NO) is a potent systemic vasodilator molecule that has a wide role in cellular systems. NO relaxes smooth muscle cells, recruits endothelial progenitor cells, promotes angiogenesis, inhibits platelet aggregation, and decreases inflammation [22]. Both EC and VSMC interact with each other to regulate the structural integrity of the vasculature. There are several growth factors and cytokines apart from NO that influence the vascular remodeling including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF) etc. [23–26]. These factors also regulate neointima formation where proliferation and migration of VSMCs to the intima is the central event [27]. ROCK is expressed in both the vascular EC and SMC [28,29]. ROCK is implicated in various cellular functions, including regulation of the actin cytoskeleton reorganization of EC and VSMC, which is associated with vascular remodeling [30,31]. ROCK regulates actin–myosin association and VSMC contraction through myosin light chain (MLC) phosphorylation and inhibits their activity [32]. Additionally, it has been shown that ROCK may also cause formation of vascular lesion formation that has been demonstrated in several models of vascular disease [33–36]. However, the role of ROCK in relation to NO bioavailability and its influence on EC, VSMC interactions and subsequent vascular remodeling is a topic of further investigation. There is a strong influence of NO on ROS production and inflammation. NO is involved in inhibition of NADPH oxidase that produces superoxide [37] and it also limits inflammation induced by other ROS such as hydrogen peroxide [38]. NO controls inflammation and thrombosis by regulating vesicle trafficking and also stimulates guanylyl cyclase and elevates a cGMP level which controls some of these effects. NO is synthesized enzymatically from L-arginine by NOS in a two-step process via the formation of the N-hydroxyl L-arginine [39]. NOS are directly or indirectly regulated by Ca2+, and which converts L-arginine into a compound, which stimulates sGC and behaves similar to endothelium

derived relaxing factor (EDRF) [40]. NOS isozymes are homodimeric, two-domain enzymes with a shared domain layout containing iron protoporphyrin IX, flavin adenine dinucleotide, flavin mono-nucleotide, and tetrahydrobiopterin, as bound prosthetic groups [41]. However, for all the three isoforms of NOS the NO synthesis depends upon the enzyme binding capacity to the calcium regulatory protein calmodulin. 3. Rho GTPases in cardiovascular health Abnormalities in the activation of Rho GTPase/ROCK pathway have been reported in various cardiovascular diseases (CVDs), such as pulmonary hypertension, cardiac hypertrophy, atherosclerosis, restenosis and myofibrillogenesis [42–46]. Air pollution exposure potentiates hypertension through reactive oxygen species-mediated activation of Rho/ ROCK [44–46]. The smooth muscle-selective RhoGAP GRAF3 is a critical regulator of vascular tone and hypertension [47,48]. The active Rho family protein has been found to promote the formation of fiber tension in vascular smooth muscle cells (VSMCs) by functioning with serotonin– threonine kinase [49,50]. Endothelial nitric oxide synthase (eNOS) is highly implicated in cardiovascular diseases and found to be vascular protective. In hypoxic condition, expression and activity of ROCK increases, which induces the downregulation of eNOS by destabilization of the eNOS transcript [51]. Rho GTPase upregulates eNOS expression mediated by HMGCoA reductase inhibitors. Since decrease in activity and expression of eNOS may lead to cardiovascular defects such as atherosclerosis in coronary artery and pulmonary hypertension, selective inhibition of endothelial Rho activity may be helpful in cardiovascular disorders [52]. Increase in eNOS expression is already reported when RhoA/ROCK signaling pathways were inhibited directly by ROCK inhibitors. Similar results were observed in the study using the dominant negative mutant of RhoA protein [53]. Takemoto et al. reported a decrease in ROCK mediated eNOS expression, when hypoxia was induced in human pulmonary EC. Rho GTPase/ROCK inhibition causes differentiation of cardiomyocytes [51], which was shown on the basis of early induction of cardiac actin expression together with up-regulated expression of the transcription factors SRF and GATA-4 [54]. Therefore, Rho GTPase/ROCKs also play an important role in myocardial differentiation. Zhao and Rivkees reported Rho GTPase/ROCK as inhibitor for cell proliferation in cultured murine embryos but it did not alter programmed cell death, which shows it plays role in cardiomyocyte division but is not involved in apoptosis of cardiomyocytes in heart development [55]. The work of Wei et al. revealed a different cardiac specific role of Rho GTPases by using transgenic expression of endogenous inhibitor, Rho GDI, of the Rho family protein [56]. They found progressive abnormalities only in AV conduction while ventricular structure and function were normal. Additionally, a critical role for ROCK-1 in mediating cardiac fibrosis has also been demonstrated [53,57,58]. Earlier work demonstrated that after 3 weeks of aortic banding, non-adaptive fibrosis was reduced in ROCK-1 knockout mice compared with wild-type mice. They also observed the induction of profibrotic gene expression, such as collagens and fibrogenic cytokines including transforming growth factor b2, was abrogated in these animals [57,58]. Another study using haploinsufficient ROCK-1 mice described substantially less cardiac fibrosis in hypertensive and infarcted ROCK-1+/– mice when compared with wild type mice thus, also concluding that ROCK-1 may be an important mediator of fibrotic heart diseases [53]. Increasing evidence shows that inhibition of the Rho/ROCK pathway may be a potential therapeutic target in CVD [59,60]. Many cellular and physiological functions are mediated by ROCK. ROCK pathway has been shown to be involved in angiogenesis [61] and other pathophysiological conditions like atherosclerosis [62], cerebral ischemia [63], hypertension [64], myocardial ischemia–reperfusion injury [43], cardiac fibrosis [58], pulmonary hypertension [65], and vascular remodeling

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[66]. Protective effects of ROCK inhibition were mediated by the acute activation of eNOS via the PI3K/Akt pathway [43]. 4. CVD drugs targeting Rho GTPases In recent years, a number of therapeutic agents, targeting Rho kinase such as Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4pyridyl) cyclohexanecarbo-xamide dihydrochloride] and fasudil, have been investigated for their efficacy in the pharmacological treatment of vascular disease such as hypertension and tubulointerstitial fibrosis [67,68]. Y-27632 is widely used as a specific inhibitor of the Rho-associated coil–coil forming protein serine/threonine kinase (ROCK) family of protein kinases. Y-27632 inhibits the kinase activity of both ROCK-I and ROCK-II in vitro, and can be reversed competitively by ATP; which suggests that Y-27632 inhibits these kinases by binding to their catalytic site. Being an inhibitor of the ROCK family of protein, Y-27632 offers a lot of therapeutic promises. Fasudil (5-(1,4-diazepane-1-sulfonyl) isoquinoline) is another important drug used worldwide targeting specifically RhoA/Rho kinase (ROCK) [69]. Mitochondrial dysfunction, in which mitochondria is unable to handle the toxic metabolites, eventually leads to myocyte injury, which has been reported to play a critical role in mediating cardiac diseases. Mitochondrial dysfunction also results in severe cardiomyopathy or myocardial dysfunction. Fasudil restored the activities of succinate dehydrogenase (SDH) and monoamine oxidase (MAO) [69] and superoxide dismutase [70] by reducing the level of RhoA, ROCK I and ROCK II proteins in in vivo. Thus, fasudil was found to inhibit the ROS and protect the mitochondria by inhibiting the mitochondrial membrane opening. It was also found to inhibit the apoptosis of cardiomyocytes due to diabetic cardiomyopathy [70]. Cardiac hypertrophy is arising due to a variety of reasons including stress in pressure or volume, mutated sarcomeric related proteins and losing of contractile mass from prior infarction and leads to cardiac dilation and functional decompensation [71]. Studies showed fasudil and its derivative hydroxyfasudil prevented the endothelin-induced cardiac hypertrophy by diminishing the hypertrophy of cardiomyocytes at micromolar (μM) level [72]. Administration of fasudil for a longer time course also resulted in hypercholesterolemic inhibition on rats including lowering of the inflammatory markers [73]. Hyperlipoproteinemia is characterized by the presence of a higher level of lipoproteins in the blood. Wang et al. [74], showed that fasudildependent inhibition of ROCK maintained the lipid metabolism at a normal level, thus protecting the cardiac system. Rho/ROCK pathway also has been shown to influence cardiac hypertrophy and vascular fibrosis through the angiotensin II-induction. Recently, fasudil has shown a concentration-dependent attenuation of angiotensin IIinduced cardiac hypertrophy without affecting the blood flow while inhibiting the pathological conditions through ROCK inhibition [74]. Hydroxyfasudil, the active metabolite of fasudil, has been shown to inhibit subarachnoid hemorrhage (SAH) and cerebral ischemia by inhibiting ROCK α and β at a concentration of 10 μM by 97.6% and 97.7%, respectively, while unaffecting other kinases by more than 40% [75,76]. This study also revealed that the selectivity of the hydroxyfasudil on the Rho inhibitors is more specific than fasudil and additionally, Satoh et al. [75,76] showed that hydroxyfasudil inhibited ROCK at 0.3– 10 μM. Thus, we believe that hydroxyfasudil would be more potent than fasudil in GTPase inhibition. Simvastatin (1S,3R,7S,8S,8aR)-8-{2-[(2R,4R)-4-hydroxy-6oxotetrahydro-2H-pyran-2-yl]ethyl}-3,7-dimethyl-1,2,3,7,8,8ahexahydronaphthalen-1-yl 2,2-dimethylbutanoate) is a type of statin, used in treating dyslipidemia and related cardiovascular diseases. It was shown that based on the pathological conditions it switches its effect on apoptosis by affecting the Rho pathway. Simvastatin may also have a direct anti-inflammatory activity by regulating ROCK and TNF-α [77]. Simvastatin induced caspase-dependent apoptosis in cardiac fibroblasts and myofibroblasts in a concentration- and time-

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dependent manner. The apoptosis of cardiac fibroblasts and myofibroblasts was affected by mevalonate, farnesylpyrophosphate and geranylgeranylpyrophosphate, greater in fibroblasts than myofibroblasts on small GTPases of the Rho family rather than Ras [78]. While studies showed that the apoptosis inhibition of simvastatin would be a cell-specific phenomenon, however based on pathological conditions, very recently, simvastatin was reported to inhibit apoptosis in ET induced cell proliferation of basilar artery by down regulating Rho/ROCK pathway in in vivo models [79]. Further studies in understanding molecular mechanisms of these phenomena would formulate simvastatin as an efficient drug for vascular remodeling associated CVD. Recent findings by Hannan and co-workers demonstrated that up-regulation of the RhoA/ROCK signaling pathway upon bilateral cavernous nerve injury has detrimental effects on erectile function. They also reported that ROCK inhibition using Y-27632 improved erectile dysfunction associated with bilateral cavernous nerve injury by preserving penile nitric oxide bioavailability and decreasing penile apoptosis [80]. Another recent study demonstrated that ROCK activation mediated by cIMP plays a critical role in hypoxic augmentation of coronary vasoconstriction. The study revealed that hypoxic augmentation of contraction of arteries with endothelium was enhanced by inosine 5′-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr(853), which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK [81]. CVD is caused by numerous molecular signaling mechanisms including reactive oxygen species (ROS), scavenger receptors and through several other mechanisms. Myristoylated alanine-rich C-kinase substrate (MARCKS) is one of the Ca2+ signaling molecule and a potential therapeutic target for CVD [82]. ROCK inhibitor HA-1077 had suppressed the spasm of cerebral arteries after subarachnoid hemorrhage. The derivative, (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]homopiperazine from H-1152P, showed specific selectivity on ROCK in protein kinase (PK) groups with very minimal kinase value as follows: 1.6, 630, 9270 nM for ROCK, protein kinase A and PKC respectively. H-1152P also suppressed the phosphorylation of MARCKS in neuronal cells and stimulated with lysophosphatidic acid, thus preventing the CVD. Some of the Rho inhibitors used in CVD are presented in Table 1 [80–100]. 5. Shear stress activates eNOS: a defining factor in cardiovascular functions The vascular influence of hemodynamic forces can be explained as: the effect of shear stress, where the endothelial

Table 1 Rho-kinase inhibitors used for CVD. S. NO

Inhibitor

Conditions

Reference

1. 2. 3.

Lovastatin Cerivastatin Y-27632

[83,84] [85] [80,81,86–89]

4. 5. 6. 7. 8. 9. 10.

H-1152P Fasudil H-1152 H-1152 Dihydrochloride GSK 269962 GSK 429286 Simvastatin

Cardiovascular diseases Cardiovascular diseases Hypertension, erectile dysfunction Hypertension Hypertension Vasoconstriction Vasoconstriction Vasodilation Vasodilation Vasodilation

[90–93] [94–96] [22,97] [37] [38] [39,40] [41]

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monolayer transduces mechanical signals into biologic responses, and circumferential deformation, leading to pathological responses. Until recently, the mechanisms of transduction by the physical force of flow into a wide range of intracellular biochemical events are an enigma. It is now recognized that the EC is the interface between the blood and the vascular wall, and an effective biological mechanotransducer. ECs are major cell types that are influenced by shear stress. There are several signal-transduction mechanisms by which shear stress exerts its physiological effects on endothelial function. Shear stress effect on EC is determined from the velocity gradient at the wall and the ability of the perfusing fluid to transmit shear forces through frictional contact. Shear stress is a hemodynamic force over the surface of the endothelium that modulates cell activities including regulation of vascular tone and inflammatory responses [101]. Previous studies showed that shear stress modulates vascular remodeling [102]. Major peripheral vascular and ischemic heart diseases are characterized by insufficient blood flow in relation to lower shear stress [103,104]. The nature and magnitude of shear stress plays a prominent role in healthy maintenance of the structure and function of the blood vessel. The nature of shear stress experienced by EC is a function of blood flow patterns throughout the vasculature. While steady shear stress generally stimulates many of the EC responses, same signaling molecules are up- or down-regulated by low and pulsatile shear stress [105,106]. Complex networks of several intracellular pathways including mechano-sensing pathways such as protein kinase C (PKC), FAK, c-Src, Rho family GTPases, PI3K, and MAPKs are triggered upon shear stimulation [107,108]. Steady shear stress activates different biochemical signals within a span of seconds, minutes to hours and days. G proteins, intracellular K+, Ca2+ concentrations [108–110], undergo a rapid increase within seconds of shear stress application [111], which further leads to multiple cellular responses. Signaling molecules like NO, prostaglandins, MAP kinases, bFGF, ICAM and other cytoskeletal signaling molecules get activated within minutes of shear stress induction [107,108]. Physiologically, the shear rate decreases at the center of the lumen and gradually increases toward the vessel wall. While the high shear magnitude protects and helps in the healthy maintenance of the EC system, areas of the arteries experience disturbed or low shear stress due to disturbed blood flow, which leads to pathological conditions such as atherosclerosis [112,113]. Shear stress has a strong influence on cytoskeletal organization and the shape of the EC from typical elongated orientation under laminar shear stress to polygonal with low shear stress [112]. Physiological shear stress is associated with eNOS expression, release of vasodilators like NO and prostacyclin [114]; antioxidative enzymes like Mn SOD and glutathione peroxidase [115] are downregulated under low shear stress conditions. Low shear stress is a crucial determining factor of apoptosis. Activation of pro-apoptotic pathways such as caspases and inflammatory factors involves increase in MAP kinase and ROCK pathways [6,116]. Other inflammatory factors include oxidative enzymes such as NADPH and xanthine oxidase; chemoattractants like MCP-1, adhesion molecules – VCAM-1, ICAM-1, ICAM-2, E-selectin, cytokines like IL-9 receptor, IL-2 receptor α, IL-3 receptor α, TNF receptor 7, integrins and IFN-γ were upregulated under low shear flow conditions [117]. Studies demonstrate that inhibition of Rho/Rho kinase increases the expression and activity of eNOS [7,118]. Some other studies show that pathological conditions such as diabetes, atherosclerosis, pulmonary and arterial hypertension etc., are associated with endothelial dysfunction, decreased NO bioavailability and increased ROCK activity [8,60]. Studies in eNOS–/– demonstrate that the effect of ROCK inhibitors is limited in relaxation of isolated corpus cavernosum (CC). However, there is a concentration-dependent effect of CC relaxation: high concentrations of Y-27632 significantly en-

hancing NO release. This study indicates that NO induces ROCK inhibition and the role played by ROCK is crucial in many vascular functions and the maintenance of its homeostasis. The nature, location, and mechanism of action of endothelial flow-sensitive mechanotransducers still remain to be explored. In addition, it is also unclear whether Rho/Rho kinase inhibition would improve eNOS/NO expression and bioavailability and thereby improve vascular functions.

6. ROCK and ROCK inhibitors perturb nitric oxide signaling: an experimental evidence Rho GTPases are implicated in a variety of physiological functions associated with changes in the actin cytoskeleton, such as cell adhesion, motility, migration, and contraction [4,119]. Increasing evidence shows that inhibition of the Rho/ROCK pathway may be beneficial in cardiovascular diseases and a potential therapeutic target [120,121]. Many cellular and physiological functions are mediated by ROCK, and elevated levels of ROCK are observed in disorders of the cardiovascular system. As discussed earlier, the ROCK pathway has been shown to be associated with several CVDs suggesting its important role in pathogenesis of the cardiovascular system. Protective effects of ROCK inhibition were mediated by the acute activation of eNOS via the PI3K/Akt pathway [43]. From these studies we can infer that inhibition of ROCK proves to be a potential therapeutic target in reducing cardiovascular disease. But how this protective mechanism of ROCK inhibition modulates angiogenesis, and what role does eNOS/NO pathway plays under shear stress need further investigation. As NO is implicated in EC migration, the question arises: By what mechanisms are shear stress, NO and migration functionally associated? We hypothesize that NO produced by EC under shear stress triggers the EC migration leading to angiogenesis. The aim of the present review is to addresses the role of ROCK in angiogenesis under shear stress, and the relation of ROCK in eNOS/NO signaling under shear stress. Endothelium, the innermost layer in blood vessels, is very specialized in the sense of detecting the changes in shear and acts according to the flow type, and shear is a major factor in determining the phenotypic and genotypic characteristics of the endothelium. Therefore, shear stress and NO play defining roles in vascular functions, and recent advances in the field of vascular biology clearly indicate that ROCK is a critical player in vascular homoeostasis particularly in the perspective of biophysical perturbations like flow modulation and shear stress. However, studying the effects of altered flow on EC in vivo is cumbersome. This problem may be best approached by studying the cells by biochemical and biophysical approaches under conditions of characterized shear stress. An in vitro apparatus has been developed based on the previous model described by Frangos et al. [106], to assess the dynamic response of endothelial cultures to physiological range fluid shear stress. In brief, endothelial shear stress (ESS) is calculated in a parallel plate flow chamber using the following formula, τ = 6 Qμ/bh2, where Q is the flow rate (cm3/s); μ is the viscosity (0.01 dyn/cm2); h is the channel height (0. 019 cm); b is the slit width (2.1 cm); and τ is the wall shear stress (dyn/cm2). The flow rate was controlled by adjusting the relative distance between the two reservoirs by changing the length of the overflow manifold tubing. Reynolds number is an important factor to determine whether the flow will be laminar or turbulent for a given geometry. For the range of shear stress used in the present study, the Reynolds number varied from 0 to 20, indicating that fluid flow through the chamber was laminar. By this shear stress model, our understanding of how these stimuli signal the cellular machinery is a key determinant in our attempts to mark diagnostic or therapeutic targets for CVD.

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6.1. Application of shear stress to EC EAhy 926 monolayers were placed in a parallel plate flow chamber and subjected to 15 dyne/cm2 shear stress for 30 min time period, in an attempt to approximate physiological conditions, as described in detail elsewhere [106]. 6.2. Estimation of NO by DAF-FM and NO electrode To study the effect of ROCK inhibitor Y-27632 on NO production Eahy926 cells grown on cover glasses were pre-treated with 0.1, 1, 5, 10 and 20 μM Y-27632 for 15 min and exposed to shear stress for 30 min, placing concurrent controls. NO was detected with a fluorescent dye DAF-FM using Varian Cary Eclipse UV–vis fluorescence spectrophotometer. NO measurement was also done by ultrasensitive NO electrode which will give the real time NO production. Treatments were given as said above then the electrode was placed nearer to the cells after 30 min of shear. 6.3. Real time NO detection using DAF-2DA To check the NO production in real time EC were treated with 5 μM of Y-27632 for 15 min prior to shear. After 30 min of shear cells were treated with DAF-2DA and washed twice with 1× PBS. Time-lapse images were taken with Olympus IX71 epifluorescence microscopy system equipped with a DP71 camera. Fluorescence intensities of the cells were calculated using image analysis module of Adobe Photoshop ver. 7.0. 6.4. Measurements of H2O2 H2O2 production was measured with amplex red fluorimetric analysis method as stated elsewhere [122]. 6.5. Measurement of peroxynitrite Production of peroxynitrite (ONOO–) was measured by using a fluorescence probe hydroxyphenyl fluorescein (HPF) as described elsewhere [123]. Briefly, ECs treated with Y27632 were applied for 30 min shear stress followed by 20 min incubation with HPF. Images were taken using an Olympus inverted fluorescence microscope at 515 nm emission. Fluorescence intensity of the cells was calculated by using image analysis module of Adobe Photoshop ver. 7.0. Our preliminary experiments carried out using Y-27632, inhibitor of ROCK on NO production showed that NO production was increased about 2 fold under 10–20 μM of Y-27632 in contrast with lower concentrations (0.1, 1 and 5 μM) reduced the NO production in similar shear condition (Fig. 1A). Similar results were obtained with ultra-sensitive NO electrode (Fig. 1B). Live cell NO production using DAF-2DA, showed that the NO hotspots that are typically seen in the peri-nuclear region of the EC with high level intensity under lower concentrations of Y-27632 were not seen under 5 μM of Y-27632 (Fig. 1C). Therefore, these set of data suggested a concentration-dependent dual effect of ROCK inhibitor on shear stress induced NO production. H2O2 production was reduced under Y-27632 by 2-fold in static, and 4-fold in shear condition. Treatment with 5 μM Y-27632 caused an increase in H2O2 production under flow conditions, particularly after 30 minutes of shear (Fig. 1D). A 2-fold increase was observed in peroxynitrite level with 5 μM Y-27632 treatments under shear compared to static controls (Fig. 1E). Interestingly cells treated with 10 μM Y-27632 showed a significant decrease in peroxynitrite level under shear compared to controls. However, no significant changes were observed in peroxynitrite levels under shear treatment alone compared to static controls. Therefore, these sets of data suggested that H2O2 is possibly acting as a sink for superoxide radicals. A concentration-dependent dual

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effect of ROCK inhibitor is evident on shear stress induced NO production. 7. Level of shear stress defines the effects of ROCKI in the endothelium: a clinically relevant issue Shear stress, a hemodynamic force, is one of the potent physiological stimuli for endothelial functions. Major peripheral vascular and ischemic heart diseases are characterized by insufficient blood flow in relation to lower shear stress [103–105,121]. Shear stress applied to EC promotes the growth of micro vessel network formation including vascular remodeling [124], which is associated with NO availability. Increased shear stress has been shown to modulate NO production [125,126] by phosphorylation of eNOS on different serine residues or dephosphorylation of T497 followed by endothelial malfunctions [127,128]. The role of Y-27632 in the range of 10 μM has been implicated in endothelial signaling pathways by inhibiting the development of atherosclerosis and arterial remodeling after vascular injury [129,130]. Rho kinase inhibitor prevented the decrease in the NO-mediated vasodilator response. In vivo inhibition of Rho kinase has been shown to preserve renal blood flow in renal ischemia–reperfusion by improving eNOS function [130]. Short-term administration of Y-27632 reduced blood pressure in various rat models of systemic hypertension [64,131]. It has also been shown that inhibition of ROCK effectively reduces pulmonary vascular resistance in an animal model of pulmonary hypertension and chronic hypoxia [65,132]. These works suggest that the ROCK pathway plays a prominent role in the pathogenesis of hypertension and its associated vascular disease [64,85,131]. Most of the works till date have widely used 10 μM Y-27632, stating the lower concentrations like 5 μM Y-27632 are toxic to the system [133]. However, its concentration-dependent roles in the endothelium have not been evaluated specifically in relation to NO dynamics under shear stress. Therefore, we propose a role of ROCK in cellular migration and angiogenesis in the presence of shear stress in relation to NO. The present work demonstrates concentration-dependent effects of Y-27632 on the cardiovascular system. A lower concentration (5 μM) of Y-27632 reverses the effects of higher concentration of the same on the endothelium. Y-27632 is being used in various experimental and clinical conditions such as cardiovascular diseases (CVD), pulmonary hypertensions, liver and renal diseases [134–136]. However, the desirable and not so desirable effects of the pharmacological treatments are highly concentration specific. We observed a concentration-dependent variable effect of ROCK inhibitor Y-27632 on NO production under shear condition. Concentrations of Y-27632, 10 and 20 μM increased NO production in EC, while 0.1–5 μM Y-27632 attenuated NO production under shear stress (Fig. 1A). This striking observation indicates that the therapeutic potential of Y-27632 could be highly concentration-dependent, and the lower concentrations of this inhibitor could be deleterious to the endothelium because of low NO production. Shear stress has been shown to activate eNOS through the activation of Akt [137]. eNOS phosphorylation at Ser-1177 by Akt therefore represents another important regulatory mechanism of eNOS activation in addition to Ca2+/CaM-dependent pathway. The work of Weber et al. [138] demonstrates that shear stress modulates eNOS mRNA stability and translation via increased 3 polyadenylation [138]. We anticipate a cross talk between NO signaling and Rho GTPases defines endothelial functions under shear stress. Recent findings establish that Rho GTPases are the key regulators of angiogenesis, and modulate diverse cellular processes including vascular permeability, extracellular matrix remodeling, migration, proliferation, morphogenesis, and survival [139–142]. Specifically the ROCK inhibitor, Y-27632, has been widely used in experimental setup, and recently in clinical practice to modulate

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Fig. 1. A. NO production under y-27632 treatment. NO level under lower concentrations (0.1, 1 and 5 μM) of Y-27632 was decreased under shear whereas the higher (10 and 20 μM) it was increased. Relative fluorescent intensity obtained was plotted in a graph. B. Corresponding data analyzed with NO sensitive electrode have been graphically presented below. * Normal vs SS (* P < 0.05); # SS vs SS + RI (#P < 0.05); † normal + RI vs SS + RI (†P < 0.05). C. Time-lapse photographs for every 5 min were taken with a DP71 digital camera attached to Olympus IX71 epifluorescence microscope shows the higher intensity of NO near the perinuclear region. D. H2O2 levels under static, shear stress conditions (5 min and 30 min) with Y-27632 (5 μM). * SS-5 min vs SS-30 min and # SS-30 min vs SS-30 min-RI. *# P < 0.05. E. Graph representing relative fluorescent intensity of peroxynitrite (ONOO–) levels under shear stress and Y-27632(5 μM). * Static vs Static 5RI, Static 10RI, # SS vs SS + 5RI, SS + 10RI; *# P < 0.05.

the cytoskeleton pattern, and thereby migration of the cells [53,143]. It is evident that EC reorients in response to shear stress by a twostep process involving Rho-induced depolarization, followed by Rho/ Rac-mediated polarization and migration in the direction of flow [144]. Ming et al. [118] demonstrated that an external stimulus like thrombin activates Rho/ROCK pathway leads to dephosphorylation of eNOS, while inhibition of Rho/ROCK reverses the effect [118]. Under static conditions RhoA/ROCK inversely regulates eNOS expression through alteration in eNOS mRNA stability [51]. We speculate that Rho-GTP regulates vascular network remodeling via NO signaling under shear stress. The work of van Nieuw Amerongen et al. [145] demonstrated a clear concentration dependent effect of Y-27632 on VEGF induced EC migration that extends a support to our observations. Uchida et al. has shown that 100 μM of Y-27632 has an anti-migratory property which could be used with other antiangiogenic compounds as an anti-angiogenic drug [146]. However,

the dosage they have used is higher compared to the mostly used dosage, which is 10 μM. On contrary to the observation of Uchida et al. [146], Noma et al. [147] demonstrated that Y-27632 (10 μM) has pro-angiogenic effects [147]. This paradox indicates that Y-27632 possibly works in a concentration-dependent manner. As Rho is implicated in the inhibition of the AKT/PKB pathway, which is a prime eNOS activation pathway [118], we deem that RhoA converges on the AKT/PKB pathway to modulate the NO production under shear stress, and thereby implicating in the ring formation. Additionally, a recent finding by Fujita et al. demonstrated that NO activated Rho-associated kinase along with activation of the PI3K–AKT signaling pathway, which leads to greater migration and invasion of pancreatic cancer cells [148]. There are also reports describing long-term exposure of NO induced lung cancer cell migration is possible via different signaling pathways including ROCK signaling [149].

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NO and downstream signaling activator cGMP regulates important functions in many vascular EC and smooth muscle cells (VSMCs) [150,151]. It is known that phosphodiesterase type 5 inhibitors (PDE5) are the most widely used drugs for erectile dysfunction and also improve lower urinary tract symptoms, a condition due to a result of an increased RhoA/ROCK signaling. Morelli et al. showed that the ROCK inhibitor Y-27632 induces relaxation in bladder strips from hypertensive rats [152]. Mills et al. have shown that a cGMP dependent modulation of Y-27632 leads to penile erection in rats, in a pathway independent of NO/cGMP and might be of use in the therapy of erectile dysfunction [153]. These observations improvise the logical next step would be to dissect the cGMP pathway to understand if ROCK inhibition and its implications in NO signaling are cGMP dependent or independent. The correct intracellular localization of eNOS is crucial for its activity [154]. We observed that Y-27632 in combination with shear stress, induced re-localization of phospho-eNOS from membrane to nuclear region (data are not shown) that possibly reduced NO production. Iwakiri et al. showed that nuclear localization of eNOS by using nuclear-localized eNOS fusion proteins with nuclear localization signal compromises NO production [155]. Another work of Klinz et al. revealed that eNOS phosphorylated at Ser-114, a negative modulation of eNOS, is heavily enriched in the nucleus, whereas eNOS phosphorylated at Ser-1177 is localized at filamentous structures in the cytosol that are abundant in the perinuclear region [156]. We postulate that sub-cellular localization of RhoA in static state is possibly different from that of under shear stress, which ultimately defines the endothelial functions in relation to NO production. It has been shown that active RhoA translocates to the membrane in the EC of the aorta of L-NAME induced hypertensive rats without changing RhoA expression level [157]. Based on these observations and our experimental evidence we deem that shear stress promotes sub-cellular translocation of Rho Kinase to remote membrane compartments, where Y-27632 specifically at lower concentrations does not get hold of Rho kinase. This erroneous localization of the enzyme possibly allows Rho kinase to be in active state. Vice versa translocation of phosphorylated eNOS to perinuclear or nuclear locations under shear stress with Y-27632 treatments destabilizes RhoA–eNOS functional relationship that could be the plausible explanation of lesser production of NO with the Y-27632 treatments under shear stress. It was observed from earlier studies [146,158–160] that there are varied effects of the ROCK inhibitor depending on the conditions and the dosage that would either enhance or inhibit its action in the vascular system. Studies in mice demonstrate that acute administration of ROCK inhibitor decreases I/R injury of the endothelium; however, this effect is nullified in eNOS–/– mice [43,158]. Similarly, ROCK inhibitor fasudil reduces myocardial infarction in occluded rats. But these cardiovascular protective effects of fasudil are mediated by PI3-kinase and eNOS [43]. These studies indicate that vascular protection of ROCK is not only a concentrationdependent effect but also dependent upon its co-inducers such as PI3-kinase, Akt and eNOS [43,158,161]. These findings suggest that ROCK may play an important role in mediating the inflammatory response to I/R injury. Shear stress is one of the key biophysical factors influencing the bioavailability and delivery of a drug. For therapeutic dosage of drugs including ROCK inhibitors, levels of shear magnitude and enhancing factors such as NO should be considered. To summarize, this review evaluates the concept that ROCK inhibitor Y-27632 perturbs endothelial functions variable under shear stress in a concentration-dependent manner. A combination of shear stress and lower concentrations of Y-27632 interferes with NO signaling. Y-27632 under low concentrations inhibited eNOS activation in EC (Fig. 2). However, these observations are cell-based studies and require further validation using animal models. In this review we

7

Static

Flow Low concentration of ROCKI

ROCK

Nitric Oxide

+

Vascular Functions

-

Fig. 2. A low dose of ROCK inhibitor (Y-27632) exerts differential effect on vascular functions specifically on endothelium based on the flow condition of the micro milieu. A laminar shear flow and low concentration of ROCK inhibitor in combination deteriorates endothelial functions.

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