Rac regulates cardiovascular superoxide through diverse molecular interactions: more than a binary GTP switch

David Gregg, Frederick M. Rauscher, and Pascal J. Goldschmidt-Clermont

Department of Medicine, Division of Cardiology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
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 ABSTRACT
 BIOCHEMICAL STRUCTURE AND...
 RAC AND NADPH OXIDASE...
 RAC ACTIVATION BY SURFACE...
 RAC ACTIVATION BY RECEPTOR...
 RAC ACTIVATION BY ANG...
 RAC IN CARDIOVASCULAR DISEASE
 FUTURE
 REFERENCES
 
The small G protein Rac has been implicated in multiple cardiovascular processes. Rac has two major functions: 1) it regulates the organization of the actin cytoskeleton, and 2) it controls the activity of the key enzyme complex NADPH oxidase to control superoxide production in both phagocytes and nonphagocytic cells. In phagocytes, superoxide derived from NADPH has a bactericidal function, whereas Rac-derived superoxide in the cardiovascular system has a diverse array of functions that have recently been a subject of intense interest. Rac is differentially activated by cellular receptors coupled to distinct Rac-activating adapter molecules, with each leading to pathway-specific arrays of downstream effects. Thus it may be important to investigate not just whether Rac is activated but also where, how, and for what effector. An understanding of the biochemical functions of Rac and its effectors lays the groundwork for a dissection of the exact array of effects produced by Rac in common cardiovascular processes, including cardiac and vascular hypertrophy, hypertension, leukocyte migration, platelet biology, and atherosclerosis. In addition, investigation of the spatiotemporal regulation of both Rac activation and consequent superoxide generation may produce new insights into the development of targeted antioxidant therapies for cardiovascular disease and enhance our understanding of important cardiovascular drugs, including angiotensin II antagonists and statins, that may depend on Rac modulation for their effect.

small G proteins; antioxidants; atherosclerosis; NADPH oxidase


THE SMALL GTP-BINDING PROTEIN Rac is an important molecular switch integrating diverse stimuli in the cardiovascular system and transducing key signaling functions such as superoxide production (2, 25), cytoskeletal organization (73, 74), and gene expression essential for cellular proliferation and hypertrophy (21, 59). Many investigations, including recent animal models, have placed Rac as a central mediator in cardiovascular physiology, including vascular reactivity and blood pressure regulation (62), as well as in pathological processes such as cardiac hypertrophy (87), vascular hypertrophy (53, 81), leukocyte migration (42), and platelet activation (35, 85) (Fig. 1). This review highlights the importance of Rac-mediated signaling in the cardiovascular system and particularly the vascular wall, with emphasis on the role of Rac as a regulator of the NADPH complex and the ability of Rac to be differentially activated by diverse mechanisms.



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Fig. 1. Rac in the cardiovascular system. The small G protein Rac regulates a diverse array of cellular and physiological processes in all major cell types of the cardiovascular system. PLT, platelets; IEL, internal elastic lamina; EEL, external elastic lamina; SMC, smooth muscle cells. Numbers following labels indicate references.

 

The role of Rac as a regulator of the NADPH oxidase complex was first described in phagocytes, where the isoforms Rac1 and Rac2 control respiratory burst oxidation. Recently, an NADPH oxidase complex, regulated by Rac1, has been characterized in nonphagocytic cells, such as vascular smooth muscle, cardiac myocytes, and endothelial cells (for review, see Ref. 32). Rac-regulated production of superoxide by the nonphagocytic NADPH oxidase has proved to be a core element in the transduction of cardiovascular signals including angiotensin II (ANG II) (77, 81), PDGF (74), thrombin (85), endothelin (18), and leukotriene B4 (LTB4) (108) (Table 1). In contrast to phagocytes, nonphagocytic cells exhibit low-intensity basal production of superoxide, tightly controlled by Rac1, as well as small bursts of NADPH oxidase activity upon Rac1 stimulation. The control of redox-dependent cellular events represents a relatively new paradigm in cell signaling that has been recently reviewed (30, 49). We will highlight the importance of examining superoxide regulation by Rac, because the cross talk and spatial confinement that it confers may help explain the complex and sometimes contradictory finding with oxidant modulation.


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Table 1. Cardiovascular signal involving Rac

 


    BIOCHEMICAL STRUCTURE AND FUNCTION
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 ABSTRACT
 BIOCHEMICAL STRUCTURE AND...
 RAC AND NADPH OXIDASE...
 RAC ACTIVATION BY SURFACE...
 RAC ACTIVATION BY RECEPTOR...
 RAC ACTIVATION BY ANG...
 RAC IN CARDIOVASCULAR DISEASE
 FUTURE
 REFERENCES
 
Rac is a member of the Rho (Ras homology) family of small (20-40 kDa) monomeric GTP-binding proteins (small G proteins), all of which undergo regulatory control by binding GTP for activation and hydrolysis to GDP for inactivation. In all known cases, the interaction of Rac with effector molecules requires that Rac be bound to GTP; thus Rac "activity" is traditionally viewed as being synonymous with GTP binding. Although Rac may mediate multiple stimuli originating both in the cytosol and at the plasma membrane, GTP-binding appears to be a common pathway of Rac activation. Binding of GTP is constitutively inhibited by guanine dissociation inhibitors (GDIs) and is enhanced by guanine nucleotide exchange factors (GEFs), whereas hydrolysis to GDP is promoted by GTPase-activating proteins (GAPs), which activate Rac's intrinsic GTPase activity (Fig. 2). GEFs for Rac, which often share binding with other small G proteins, comprise a growing group of proteins containing tandem DH (Dbl homology) and PH (pleckstrin homology) domains. When a PH domain undergoes phosphorylation or interaction with membrane-associated phosphatidyl inositides [such as phosphatidylinositol 3,4,5-trisphosphate (PIP3)], GEF activity, catalyzed by the proximate DH domain, is enhanced (33). Of the eight GEF proteins that activate Rac in vitro (Sos1, Vav1-3, Trio, Ost, Bcr, Abr, Ect2, and Tiam1), all but Tiam1 have so far been shown to activate other small G proteins, particularly Ras, which helps explain frequently observed intracellular small G protein "cross talk" (80, 98).



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Fig. 2. Rac regulation by GTP cycling. Inactive Rac is bound to GDP and constitutively inhibited by guanine dissociation inhibitors (GDIs) to prevent unsignaled GTP exchange. Once an agonist signals Rac activation, guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP and membrane localization. GTPase-activating proteins (GAPs) then regulate the hydrolysis of GTP back to inactive GDP-bound Rac. P, phosphate.

 

Individual GEFs, by possessing distinct lipid- and protein-binding motifs in addition to sites to bind Rac, seem to dictate which subset of a multitude of downstream effectors are targeted upon Rac activation (13, 23). For example, whereas GEF-independent activation of Rac stimulates both p21-activated kinase (PAK) and c-Jun NH2-terminal kinase (JNK) pathways, the Rac-specific GEF Tiam1 preferentially activates PAK, with minimal JNK activation (115). This bears physiological significance because PAK and JNK mediate distinct cellular events (cytoskeletal reorganization and gene expression, respectively). Thus, although Rac is always activated through a common pathway of GTP binding, the GEFs potentially allow diverse upstream signals to culminate in downstream cellular events that are equally diverse and specific.

Once bound to GTP, Rac is self-inactivated through slow intrinsic GTPase activity, which is greatly enhanced by interaction with GAPs. The enhancement is through a conserved GAP domain, found for example in N- and {beta}-chimerins, that inserts into the active site of Rac to stabilize the transition state and thus favors GTP hydrolysis (10). In addition to regulating the speed of GTP-GDP cycling, individual GAPs also contain unique domains that mediate downstream effects. In fibroblasts, microinjection of N- and {beta}-chimerins, including mutants lacking GAP activity, induces Rac-dependent lamellipodia formation (46). In this sense, GAPs may be viewed as a special set of Rac effector proteins, not only capable of binding to activated Rac to transduce downstream signals but with the additional ability to turn off their own upstream activation. Coordinated cell function may also be facilitated by the ability of GAPs to inactivate multiple members of the small G protein family, which occurs with p190 RhoGap. Moreover, phosphorylated p190 can bind to the SH2 domain of the Ras-specific GAP p150, producing cross talk between effector proteins (82). In addition, more complex molecules such as Bcr can exhibit both GEF activity for one small G protein and GAP activity for another, allowing it to activate one pathway while silencing another (14).

In addition to domains for binding and hydrolyzing GTP, Rac activity also requires a COOH-terminal geranylgeranyl moiety. This modification, essential for the localization of Rac to the plasma membrane upon activation (73, 90), arises from posttranslational addition by the enzyme geranylgeranyl transferase (GGT) (12). Interestingly, geranylgeranyl groups, like cholesterol, are derived from the mevalonate pathway, and their production is blocked by HMG-CoA reductase inhibitors (statins) (73, 90). Thus modification of small G proteins by statins may contribute to their beneficial effect in cardiovascular disease (91). The geranylgeranyl moiety of inactive Rac can be sequestered within the hydrophobic pockets of GDIs. These cytosolic proteins stabilize both GDP- and GTP-bound Rac to prevent unnecessary GTP cycling and inhibit inactive Rac from interacting with effector proteins, such as PAK, through concealment of key structural regions (24). Live cell imaging has revealed that proper cellular localization of Rac is crucial because certain effector molecules, such as PAK, bind to Rac only after successful membrane targeting (47). However, GDI-mediated presentation of Rac to other effectors, such as the p67phox component of the NADPH oxidase complex, appears to occur in the cytosol (17, 22).


    RAC AND NADPH OXIDASE REGULATION
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 ABSTRACT
 BIOCHEMICAL STRUCTURE AND...
 RAC AND NADPH OXIDASE...
 RAC ACTIVATION BY SURFACE...
 RAC ACTIVATION BY RECEPTOR...
 RAC ACTIVATION BY ANG...
 RAC IN CARDIOVASCULAR DISEASE
 FUTURE
 REFERENCES
 
Once activated, Rac is able to complex with its effectors to initiate downstream cellular events including NADPH oxidase, the predominant source of superoxide in both phagocytic and nonphagocytic cells and a central player in redox-dependent signaling (32, 40). Whereas Rac regulation of the NADPH has been observed in the homologous nonphagocytic oxidase of the cardiovascular system (86), as well as in phagocytes where it was first described, most molecular details of oxidase function are derived from leukocytes with extrapolation to the nonphagocytic oxidase. The NADPH oxidase is composed of four functional components whose assembly requires the presence of Rac at the plasma membrane. Two components are constitutively located in the membrane and comprise the cytochrome: Gp91phox (or its homologue in vascular smooth muscle cells, Nox1) and p22phox, a second smaller membrane subunit. The other components, p47phox and p67phox, are cytosolic adapter proteins, which complex with Rac to regulate oxidase activity. Although this model provides a general guideline to understand oxidase activation in phagocytes, important differences in molecular structure and function exist and need to be further explored in different cell types. For example, in vascular endothelial cells, gp91phox and p22phox have been recently observed as preassembled complexes in the cytoplasm rather than membrane localized (55), and smooth muscle cells do not express the adapter protein p67phox.

Interestingly, the affinity of Rac for the oxidase differs depending on which GEF is bound to Rac. In COS cells engineered to express the oxidase components Gp91phox, p22phox, p47phox, and p67phox, a constitutively active Vav1 GEF mutant exhibits greater superoxide production than active Vav2 or Tiam1 mutants, despite greater PAK binding by the Vav2 and Tiam1 mutant (68) (Fig. 3). The importance of Rac GEFs in differentially regulating the activation of the oxidase in human disease is illustrated in oncogenic transformation, where the same NH2-terminal deletion of Vav1 used to create constitutively active mutants acts as a protooncogene (43). Inherited defects in Rho family GEFs have been identified recently in faciogenital dysplasia and X-linked mental retardation (48, 63). Although a cardiovascular disease related to polymorphism in Rac or Rac GEFs has yet to be identified, subtle genetic variations resulting from Rac and Rac GEF polymorphisms may potentially shift the Rac balance between different effectors to impact predisposition to cardiovascular disease.



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Fig. 3. Differential Rac activation. Receptor binding may lead to selective activation of Rac for its different downstream effectors conferred by GEFs. Vav1 preferentially stimulates superoxide production, whereas Tiam1-regulated activation favors p21-activated kinase (PAK) binding. This bears physiological significance because PAK and superoxide may mediate distinct cellular events such as cytoskeletal reorganization and inflammatory gene expression.

 


    RAC ACTIVATION BY SURFACE RECEPTORS
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 RAC AND NADPH OXIDASE...
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 RAC IN CARDIOVASCULAR DISEASE
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The specificity of activation conferred by GEF mutants (68, 115) lays the conceptual groundwork for an understanding of differential activation by surface receptor-coupled agonists. Each receptor may initiate a different signaling pathway to Rac activation, with different molecular signals and kinetics presumably resulting in unique physiological effects. Although the full details and complexity of the activation of Rac by different agonists are still missing, hints of how it differs depending on the stimulus with respect to time course, mechanism of activity, and time to inactivation are emerging. For example, assays of Rac binding to its effector protein PAK show brisk and transient activation by Gi stimuli such as LTB4 that peaks within 2 min and no longer binds PAK at 5 min (77). In contrast, the more complex activation through the Gi- and Gq-coupled receptor ANG II also rapidly activates Rac but is sustained through 30 min (81). Studies with inhibitors of various signaling cascades have also revealed that different signaling pathway requirements are agonist dependent. For example, LTB4 activation of Rac in neutrophils is via Gi-coupled receptors that can be inhibited by pertussis toxin and requires phosphatidylinositol 3-kinase (PI3K) but not PKC (4), whereas thrombin-induced Rac activation appears independent of PI3K (85). Rac's supporting cast of GAPs, GEFs, and GDIs are likely to contribute to these unique modes of activation and regulate a balance among Rac's affinities for its multiple effectors.


    RAC ACTIVATION BY RECEPTOR TYROSINE KINASES
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The richness of signaling that may be afforded by the supporting cast of GEFs and GAPs is best illustrated by the well-characterized pathway of Rac activation by receptor tyrosine kinases (RTK) (Fig. 4). When an agonist, such as PDGF, EGF, or insulin, activates a RTK, the receptor oligomerizes, leading to autophosphorylation of a key tyrosine residue. The SH2 domain of an adapter protein, such as Grb2, recognizes this receptor change, binds the receptor, and is itself phosphorylated. This allows for a signaling complex assembly comprising E3b1, Eps8, and son of sevenless (Sos). This in turn confers GEF function to Sos, which catalyzes guanine nucleotide exchange for both Rac and Ras (11, 27, 83). The key event in GEF activation appears to be the adapter protein localizing the GEF to the membrane (6, 70). Initial evidence showed that once this complex was formed and localized to the membrane, it activated Ras, which in turn activated Rac (6, 78). The specifics of this activation of Rac through Ras have remained unclear, but it requires PI3K, whose product PIP3 is capable of activating the Rac-specific GEF Vav (33). New evidence, however, has now emerged showing both in vivo and in vitro that the Sos-Eps8-E3b1 complex can interact directly with Rac through the interaction of Eps8 with Rac. Eps8 may also contribute to proper Rac subcellular localization through the interaction of Eps8 with F-actin (39, 79). It thus appears that RTK activation of Rac can be activated directly through Sos or through a more complex Ras-dependent pathway; what still remains unclear is how these multiple pathways of activation may enhance the complexity of Rac signaling with each likely resulting in a slightly different affinity of Rac for downstream effector proteins.



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Fig. 4. Rac activation by receptor tyrosine kinase (RTK). Once an agonist, such as PDGF or EGF, binds an RTK, the receptor oligomerizes and is autophosphorylated. The adapter molecule Grb2 recognizes the receptor change and signals the assembly of the E3b1-Eps8-Sos GEF complex. Sos then catalyzes Rac's exchange of GDP for GTP to become active. A complementary activation pathway proceeds through Sos activation of Ras, which then activates Rac through a pathway requiring phosphatidylinositol 3-kinase (PI3K) and, likely, the Rac GEF Vav. GAPs then regulate the inactivation of Rac through the hydrolysis of GTP.

 


    RAC ACTIVATION BY ANG II
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 RAC AND NADPH OXIDASE...
 RAC ACTIVATION BY SURFACE...
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 RAC IN CARDIOVASCULAR DISEASE
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The molecular details of ANG II activation of Rac have also been of intense interest, and the requirement of Rac to mediate many well-characterized physiological consequences of ANG II signaling has been defined. ANG II-induced gene expression and hypertrophy can be blocked with a dominant negative Rac (92). Furthermore, ANG II-induced superoxide is inhibited by a dominant negative Rac or Clostridium difficile toxin A, a Rac inhibitor (81). Similarly, inhibiting Rac-regulated superoxide production with antioxidants or antisense to the p22phox component of the oxidase blocks ANG II signal transduction (31, 71, 97). ANG II is also capable of inducing PAK binding by Rac (77).

ANG II signaling proceeds through a heterotrimeric G protein-coupled receptor through both a Gq/11 pathway and a Gi pathway (94) (Fig. 5). With two parallel pathways resulting in Rac activation through the same receptor, investigations attempting to define the signaling requirements of each pathway have been complicated by the possibility of redundant pathways. Subtle differences in agonist dosing and kinetics also appear to produce different effects and may explain the seemingly contradictory investigations exploring the signal transduction pathways seen by Schmitz et al. (77) in contrast to Seshiah et al. (81). The most comprehensive model of ANG II activation of Rac and superoxide derives from work by Ushio-Fukai et al. (96) and Seshiah et al. (81). They propose that AT1 receptor binding leads to initial PKC activation, which directly results in rapid early reactive oxygen species (ROS) production. Sustained superoxide then proceeds through transactivation of the epidermal growth factor receptor (EGFR) by a Src kinase activated by the initial ROS burst. EGF subsequently activates PI3K with resultant activation of the small GTP-binding protein Rac, a key regulator of the NADPH oxidase complex, to result in long-term superoxide production. This model is well supported by their data, showing that inhibition of PKC with GF-109203x significantly blocks hydrogen peroxide production. Additionally, Src kinase appears to be activated by superoxide to transactivate the EGFR, because antioxidant treatment with diphenylene iodonium, an inhibitor of flavin oxidases, tiron, a superoxide scavenger, N-acetylcysteine, or ebselen, a glutathione peroxidase mimetic, will block both ANG II-induced EGFR and Src activation, whereas hydrogen peroxide treatment or treatment with the superoxide-releasing compound LY-83583 will mimic ANG II-induced EGFR phosphorylation. Similarly, inhibition of Src with protein phosphatase 1 (PP1) blocks EGFR activation and limits Rac-PAK binding and superoxide production. Further supporting this model, inhibition of EGFR kinase with AG-1478 or PI3K inhibition with wortmannin attenuated Rac-PAK binding (81, 96). In contrast, Schmitz et al. (77) found that ANG II induction of Rac activity as measured by PAK-PBD binding is inhibited by the global RTK inhibitor genistein but not the Src kinase inhibitor PP1, suggesting that activation requires a non-Src tyrosine kinase. Furthermore, although they found that activation also requires PKC, because downregulation of PKCs with the inhibitor phorbol 12,13-dibutyrate limits Rac-PAK binding, the low-dose PKC inhibitor GF-109203x had only minimal effect (77). The precise explanation for these apparently contradictory findings is unclear. Furthermore, Seshiah et al. (81) found that early PKC-derived superoxide precedes Rac activation by EGFR transactivation; however, whether this initial NADPH oxidase activation through PKC involves Rac as well remains unclear. In their different model system, Schmitz et al. (77) show Rac-PAK binding 15 s after stimulation, whereas EGFR transactivation has not been shown earlier than 1 min, suggesting perhaps that Rac is directly activated to effect the early superoxide burst and later activated through EGFR to perpetuate a more sustained activation.



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Fig. 5. Angiotensin II (ANG II) activation of Rac. ANG II activates Rac through transactivation of the EGF receptor (EGFR). After ANG II binds the Gq-coupled receptor, PKC is activated, which leads to early superoxide release necessary for Src activation of the EGFR. The signaling linking PKC to superoxide release is unclear and may involve transient activation of Rac; however, NADPH oxidase is required for transactivation, because it can be blocked by antioxidants. Rac is then activated through a process requiring PI3K to result in more sustained superoxide production. SO, superoxide.

 


    RAC IN CARDIOVASCULAR DISEASE
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 ABSTRACT
 BIOCHEMICAL STRUCTURE AND...
 RAC AND NADPH OXIDASE...
 RAC ACTIVATION BY SURFACE...
 RAC ACTIVATION BY RECEPTOR...
 RAC ACTIVATION BY ANG...
 RAC IN CARDIOVASCULAR DISEASE
 FUTURE
 REFERENCES
 
As a key signaling molecule in the transduction of multiple important cardiovascular agonists resulting in the production of superoxide, cellular motility necessary for chemotaxis and vessel wall repair, and gene expression controlling proliferation and hypertrophy, Rac plays a prominent role in the cardiovascular system in both health and disease. Several important themes have emerged demonstrating that Rac and ROS production are core signaling elements in a diverse spectrum of cardiovascular disease and biology as well as a likely player in the action of important cardiovascular drugs including angiotensin-converting enzymes and statins.

Cardiac hypertrophy. Myocardial hypertrophy can be an adaptive, or maladaptive, response to increased wall stress resulting from chronic hypertension or altered cardiac geometry after myocardial infarction; furthermore, excessive hypertrophy leads to impaired myocardial function and eventual heart failure. In addition to potentially promoting cytoskeletal and myofibrillar rearrangement, Rac also contributes to myocardial hypertrophy by activation of cellular growth pathways (19). Hyperactivation of growth signals underlies the oncogenic effects of Rac in mitotically competent cells (41), whereas similar activation in postmitotic cells, such as cardiomyocytes, appears to result in a hypertrophic phenotype. In investigations of this analogy, early evidence of the role of Rac in oncogenic transformation and the regulation of important pathways controlling genes involved in growth and proliferation, such as p38 mitogen-activated kinase (MAPK), JNK, Akt, and ERK, sparked interest in a possible role of Rac in cardiac hypertrophy. Similarly, Rac GEFs, such as Vav and Sos, had been observed to be involved in oncogenesis, further suggesting that Rac and its associated GEFs might be involved in cardiac hypertrophy. Subsequent work positioned Rac and superoxide as a necessary downstream signal of the small G protein Ras, a key regulator of mitogenesis and oncogenic transformation (41, 69), and in neonatal cardiomyocytes, adenoviral infection with a dominant negative form of Ras blunts the activation of MAPK, suggesting a possible relationship of Rac in cardiac hypertrophy (66). These findings were subsequently extended to directly implicate Rac when it was shown in neonatal rat cardiomyocytes that constitutively active Rac V12 induced sarcomeric reorganization and hypertrophy indistinguishable from phenylephrine-induced changes. Rac also resulted in increased expression of the embryonic gene atrial natriuretic peptide. Furthermore, dominant negative Rac N17 blocks phenylephrine-induced hypertrophic changes (67). In vitro work has continued to implicate Rac as a messenger of other important hypertrophic pathways including signal transduction downstream of ANG II, TNF-{alpha}, and EGF. Finally, a mouse model in which a constitutively active Rac V12 gene was expressed selectively in the myocardium confirms that Rac activation leads to cardiac hypertrophy in vivo. These transgenic mice show two phenotypes: a lethal dilated cardiomyopathy associated with neonatal expression, and a viable hypertrophy in young mice that resolved with age. Interestingly, excessive Rac activation did not lead to any myofibrillary derangement, and Rac V12 hearts were in fact hypercontractile (87).

Although PAK binding has been used to document increased Rac activation in cardiac hypertrophy, the downstream effector most likely mediating Rac-induced hypertrophy is superoxide produced by NADPH oxidase. By infection of fibroblasts with PAK mutants unable to bind Rac, Westwick et al. (106) showed that Rac-induced mitogenesis, transformation, and lamellipodia formation as well as activation of growth-related genes JNK, p38, and serum response factor (SRF) are PAK independent. Hinting that Rac-activated NADPH oxidase is the critical mediator of hypertrophy, EGF-induced calcium release in fibroblasts correlates with hydrogen peroxide production and can be blocked by dominant negative Rac or the antioxidant enzyme catalase (52). In addition, catalase and SOD mimetics also blunt mechanical stress-induced hypertrophy and activation of JNK and ERK in cardiomyocytes (65). These findings provide a mechanism by which Rac activation by ANG II can induce cardiac hypertrophy independent of blood pressure elevation (77), through increased superoxide generation (9). Indeed, ANG II-induced hypertrophy is blunted by the Rac inhibitor C3 exotoxin as well as dominant negative Rac and SOD (92). Furthermore, statins, in addition to their cholesterol-lowering effects, may also regulate cardiac hypertrophy by inhibiting Rac prenylation and oxidant signaling (51, 92). Treatment of rat cardiomyocytes with simvastatin in vitro blocks ANG II-induced hypertrophy and fetal gene expression, which correlates with decreased Rac activity and superoxide production. Convincingly, simvastatin also inhibits cardiac hypertrophy in rats treated with either ANG II or transaortic constriction, which is associated with a concomitant decrease in myocardial Rac activity and superoxide production in vivo (92).

Vascular hypertrophy. Vascular remodeling is a compensatory response to vascular insults such as hypertension and increased sheer stress and is implicated in the development of atherosclerosis. Evidence supporting the role of Rac in vascular hypertrophy stems from its role in regulating gene expression, controlling superoxide production, and sensing and transducing shear stress. Although the upstream signals are yet unidentified, Rac is activated by vascular shear stress, resulting in transduction of growth and hypertrophy signals. In vascular smooth muscle cells (VSMC), dominant negative Rac N17 blocks cyclic strain-induced activation of p38 MAPK and ERK (53, 54). Not surprisingly, this regulation appears to be superoxide dependent, with shear-induced MAPK activation equally blocked by antioxidants or RacDN (111). Heat shock protein expression and activation in response to both cyclic stress and heat stress also require Rac (110). As in cardiac hypertrophy, Rac is essential for ANG II-induced vascular remodeling, with ANG II Rac-dependently activating ERK and JNK pathways in VSMC (77). In addition, vascular NADPH oxidase components are upregulated in vivo upon ANG II infusion (15, 29). Furthermore, ANG II-induced vascular hypertrophy is blocked by catalase, SOD mimetics, and antisense p22phox cDNA in vitro (97, 113) or by knockout of gp91phox in mice (102).

Hypertension. There has been intense interest in the role of superoxide and oxidative stress in hypertension (114), because as both a key regulator of NADPH-derived superoxide and as a mediator of the potent vasoconstrictor ANG II, Rac is likely an important regulatory target in hypertension. Illustrating the importance of Rac-derived superoxide, angiotensin treatment results in increased superoxide production with significantly increased membrane-bound NADH and NADPH oxidase. Impaired relaxation to calcium ionophore, acetylcholine, and nitroglycerin are also seen after ANG II treatment, and these changes can be reversed with an SOD mimetic (71). Direct inhibition of the oxidase in a mouse model with a chimeric protein gp91ds-tat, which inhibits the interaction of p47phox and gp91phox, or use of p47phox knockout mice blunts the superoxide production and hypertension induced by ANG II treatment and clearly demonstrates that in vivo NADPH oxidase-produced superoxide is the essential mediator of angiotensin's hypertensive response (50, 72). Interestingly, the spontaneous hypertensive rat (SHR) shows enhanced superoxide production with increased expression of the oxidase component p22 and impaired vasodilatation to acetylcholine (29). Moreover, treating normocholesterolemia SHR with statins reduces blood pressure with a concomitant decrease in superoxide production as well as angiotensin receptor NADPH oxidase component expression (104). Mechanistically, Rac-derived superoxide production may act as an antagonist to vasodilatory nitric oxide (NO) through the rapid reaction of the two reactive species to form peroxynitrite, thus decreasing NO bioactivity (114).

Rac has now been directly implicated in hypertension in vivo through the development of a transgenic mouse model expressing constitutively active RacV12 under an {alpha}-actin promoter for smooth muscle-selective expression. These mice show hypertension compared with wild-type littermates, caused by enhanced superoxide production. Treatment of these mice with the antioxidant N-acetylcysteine results in reversal of the hypertension (Goldschmidt-Clermont, unpublished data). Similarly, Rac mediates the arteriolar constriction response to increased transmural wall pressure. In studies using isolated mouse tail arterioles, increased wall pressure has been shown to result in superoxide production and arteriolar contraction that could be attenuated with RacDN or superoxide inhibition (62).

Atherosclerosis. There has long been interest in the role of oxidative stress and antioxidants in the development of atherosclerosis. Recently, it was shown that cellular oxidative stress predicts mortality in patients with coronary artery disease (37). Smooth muscle cells from p47phox-/- knockout mice lacking a key component of the oxidase exhibit decreased proliferation and superoxide production. Furthermore, knockout of p47phox attenuates the development of atherosclerosis in ApoE-/- mice (8). As the core regulator of superoxide production through the NAD(P)H oxidase, Rac is positioned as a critical atherogenic signal. In mice, Rac appears to be directly linked to the consequences of hypercholesterolemia, which include increased NADPH-derived superoxide production, increased expression of the AT1 receptor, and impaired endothelium-dependent vasorelaxation. In addition, hypercholesterolemic mice have increased macrophage infiltration of the vasculature and enhanced plaque rupture that are attenuated with AT1 receptor antagonism (103). Similarly, in vascular injury resulting from balloon-induced carotid injury, there is a significant upregulation of the oxidase component Nox1 with associated increased superoxide production, pointing to a central role of NADPH oxidase-generated superoxide in restenosis (89). Coordinated Rac activity may also be important in smooth muscle migration from the media to intima required for the development of atherosclerosis, because both Ra N17 and, to a lesser extent, RacV12 inhibit smooth muscle migration (26). Similarly, endothelial migration and cytoskeletal changes controlling the integrity of the vascular wall are regulated by Rac and superoxide (60). Moreover, because Rac activation requires geranylgeranyl synthesis, which is inhibited by statins, Rac deactivation may account for the cholesterol-independent antiatherosclerotic effects of statins demonstrated in clinical trials (57, 91). In addition to lowering cholesterol, statins limit smooth muscle cell hypertrophy and vascular inflammation, improve vasoreactivity, stabilize plaques, and regulate platelet aggregation all through pathways that appear to be regulated by Rac and Rho family GTPases (91). Moreover, in VSMC, atorvastatin blocks ANG II-induced Rac activation and downstream superoxide production, an effect that is mimicked by GGT inhibitors and reversed by mevalonate (105). Interestingly, in contrast, treatment of endothelial cells with cerivastatin increases Rac activation as measured by Rac-PAK binding in total cell lysates, although statin treatment decreases the fraction of Rac that was membrane localized (100).

Atherosclerotic vessels also demonstrate reduced levels of NO, which may be mediated by NO inactivation by superoxide to produce peroxynitrite. In vivo investigations in hypercholesterolemic rabbits demonstrate statin inhibition of Rac and superoxide with resultant restoration of NO levels and plaque stabilization (93). Moreover, Rac activity enhances the expression of plaque-destabilizing cytokines such as IL-6 and monocyte chemoattractant protein (MCP-1) through superoxide (28, 56). In fact, Rac-dependent MCP-1 expression is blocked by NO, suggesting an inactivation of Rac-derived superoxide by NO (109).

Leukocyte migration, chemotaxis, and platelet aggregation. Communication of the vasculature with both leukocytes and platelets is crucial in the vascular flux of inflammatory cells leading to both chronic atherosclerosis and acute thrombus formation. Rac signaling is central to leukocyte attraction, migration, and vascular attachment via production of MCP-1, regulation of cellular motility, and expression of adhesion molecules. In addition to shear stress-induced endothelial injury that summons leukocytes, Rac also contributes to the cytoskeletal rearrangement necessary for the movement of inflammatory cells through the endothelium (42, 76). Both dominantly negative and constitutively active Rac inhibit macrophage migration (5, 42) and PDGF-induced chemotaxis of fibroblasts and VSMC. The ability of both the dominant negative and constitutively active Rac to inhibit these processes indicates that a balanced, coordinated regulation of Rac is necessary to regulate cell movement. The potent chemoattractant LTB4 also activates Rac and ROS production (108), and blockade of the leukocyte LTB4 receptor attenuates atherosclerosis in mice by diminishing monocytic infiltration of the vasculature (3).

In platelets, thrombin signaling and interaction with adhesion molecules requires Rac and contributes to subsequent thrombus formation (85). Cytoskeletal reorganization is necessary for platelet activation and morphological changes (34, 35). Rac and PAK are activated in the process of platelet spreading on collagen through a pathway that requires Src kinase and, to a lesser extent, PI3K. This appears to be independent of integrin {alpha}2{beta}1, because surfaces coated with anti-{alpha}2{beta}1 also induce Rac binding of PAK with a requirement of Src and PI3K (88). Through the use of PAK binding assays, thrombin, which signals through a heterotrimeric G protein coupled receptor, and collagen, which signals through an RTK, have now both been demonstrated to rapidly activate Rac with the requirement of phospholipase C and calcium mobilization. Activation by collagen also requires PI3K, whereas thrombin is PI3K independent (85). Interestingly, integrins appear to predominantly regulate Rac activation not through effects on GTP loading but, rather, through the plasma membrane localization of Rac that is already GTP bound. Transfection of cells with V12 Rac does not result in global PAK binding but, rather, is limited to specific plasma membrane locales where integrin activation is occurring. Thus integrins provide spatiotemporal regulation of Rac's binding to effectors, which appears to be mediated by GDI release (24).


    FUTURE
 TOP
 ABSTRACT
 BIOCHEMICAL STRUCTURE AND...
 RAC AND NADPH OXIDASE...
 RAC ACTIVATION BY SURFACE...
 RAC ACTIVATION BY RECEPTOR...
 RAC ACTIVATION BY ANG...
 RAC IN CARDIOVASCULAR DISEASE
 FUTURE
 REFERENCES
 
Knowledge and implication of Rac and its regulation of superoxide in the cardiovascular system has burgeoned over the last decade since the discovery that Rac regulates NADPH oxidase. Rac has been clearly defined to be a regulator in a diverse array of cardiovascular conditions initiating largely redox-dependent signaling cascades to mediate adaptive and maladaptive responses to stress such as hypertrophy, vasomotor tone, platelet aggregation, and leukocyte migration into the vasculature. Rac inhibition is also likely to be a major mechanism leading to some of the clinical benefits of both statins and anti-angiotensin drugs. Rac regulation, however, is much more complex than a simple binary GTP switch. Most research has defined whether Rac is involved in disease processes by demonstrating whether the switch was on or off, but often without questioning "on or off where and for what effectors?" The supporting cast of Rac GEFs, GAPs, and GDIs, through their unique effector domains, modulates the affinity of Rac for effectors and facilitates intracellular cross talk. Whereas oxidant production under Rac regulation is central to the development of multiple pathological states, the clinical efficacy of antioxidant drugs for cardiovascular diseases has been disappointing (1, 112). An understanding of Rac-activated oxidant production helps explain the frequently observed ineffectiveness of global antioxidant therapy. In contrast to respiratory burst oxidation in inflammatory cells, vascular superoxide production is mostly involved in the alteration of local redox conditions that influence the propagation of molecular signaling pathways (30, 49). Because of the inherent short half-life of free radical second messengers, cells have evolved necessary mechanisms for localizing oxidant production. Similar to the localization of NO signaling afforded by the spatial distribution of NO synthase isoforms, Rac likely mediates cardiovascular physiology through a spatiotemporal regulation of superoxide production (7). Thus, for an antioxidant therapy to work, it must be targeted to the desired physiology and cellular locale. Rac GEFs and GAPs as well as other pathway-specific signaling molecules resulting in Rac activation are more likely to be successful drug targets, because they would control a subset of redox-dependent signals rather than alter the global cell redox state. To achieve this goal, future investigations should aim to expand our understanding of how Rac-associated molecules modulate downstream binding affinities to result in varied temporal and spatial regulation and differentially regulated physiological processes. As we head into the future of molecular medicine, the next frontier in Rac signaling shall be identifying and targeting the subsets of Rac effectors that differentially activate each process.


    DISCLOSURES
 
This work has been supported by National Heart, Lung, and Blood Institute Grant R01-HL-071536-06 (to Pascal J. Goldschmidt-Clermont).


Address for reprint requests and other correspondence: P. J. Gold-schmidt-Clermont, Dept. of Medicine, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: golds017{at}mc.duke.edu).


    REFERENCES
 TOP
 ABSTRACT
 BIOCHEMICAL STRUCTURE AND...
 RAC AND NADPH OXIDASE...
 RAC ACTIVATION BY SURFACE...
 RAC ACTIVATION BY RECEPTOR...
 RAC ACTIVATION BY ANG...
 RAC IN CARDIOVASCULAR DISEASE
 FUTURE
 REFERENCES
 
1. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico. Lancet 354: 447-455, 1999.[ISI][Medline]

2. Abo A, Pick E, Hall A, Totty N, Teahan CG, and Segal AW. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature 353: 668-670, 1991, 1999

3. Aiello RJ, Bourassa PA, Lindsey S, Weng W, Freeman A, and Showell HJ. Leukotriene B4 receptor antagonism reduces monocytic foam cells in mice. Arterioscler Thromb Vasc Biol 22: 443-449, 2002.[Abstract/Free Full Text]

4. Akasaki T, Koga H, and Sumimoto H. Phosphoinositide 3-kinase-dependent and -independent activation of the small GTPase Rac2 in human neutrophils. J Biol Chem 274: 18055-18059, 1999.[Abstract/Free Full Text]

5. Allen WE, Jones GE, Pollard JW, and Ridley AJ. Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. J Cell Sci 110: 707-720, 1997.[Abstract/Free Full Text]

6. Aronheim A, Engelberg D, Li N, al Alawi N, Schlessinger J, and Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell 78: 949-961, 1994.[ISI][Medline]

7. Barouch LA, Harrison RW, Skaf MW, Rosas GO, Cappola TP, Kobeissi ZA, Hobai IA, Lemmon CA, Burnett AL, O'Rourke B, Rodriguez ER, Huang PL, Lima JA, Berkowitz DE, and Hare JM. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature 416: 337-339, 2002.[ISI][Medline]

8. Barry-Lane PA, Patterson C, van der MM, Hu Z, Holland SM, Yeh ET, and Runge MS. p47phox is required for atherosclerotic lesion progression in ApoE(-/-) mice. J Clin Invest 108: 1513-1522, 2001.[Abstract/Free Full Text]

9. Bendall JK, Cave AC, Heymes C, Gall N, and Shah AM. Pivotal role of a gp91(phox)-containing NADPH oxidase in angiotensin II-induced cardiac hypertrophy in mice. Circulation 105: 293-296, 2002.[Abstract/Free Full Text]

10. Bishop AL and Hall A. Rho GTPases and their effector proteins. Biochem J 348: 241-255, 2000.[ISI][Medline]

11. Buday L and Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 73: 611-620, 1993.[ISI][Medline]

12. Casey PJ and Seabra MC. Protein prenyltransferases. J Biol Chem 271: 5289-5292, 1996.[Free Full Text]

13. Cerione RA and Zheng Y. The Dbl family of oncogenes. Curr Opin Cell Biol 8: 216-222, 1996.[ISI][Medline]

14. Chuang T, Xu X, Kaartinen V, Heisterkamp N, Groffen J, and Bokoch GM. Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family. Proc Natl Acad Sci USA 92: 10282-10286, 1995.[Abstract]

15. Cifuentes ME, Rey FE, Carretero OA, and Pagano PJ. Upregulation of p67phox and gp91phox in aortas from angiotensin II-infused mice. Am J Physiol Heart Circ Physiol 279: H2234-H2240, 2000.[Abstract/Free Full Text]

16. Clark EA, King WG, Brugge JS, Symons M, and Hynes RO. Integrin-mediated signals regulated by members of the rho family of GTPases. J Cell Biol 142: 573-586, 1998.[Abstract/Free Full Text]

17. Clark RA, Volpp BD, Leidal KG, and Nauseef WM. Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J Clin Invest 85: 714-721, 1990.[ISI][Medline]

18. Clerk A, Pham FH, Fuller SJ, Sahai E, Aktories K, Marais R, Marshall C, and Sugden PH. Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1. Mol Cell Biol 21: 1173-1184, 2001.[Abstract/Free Full Text]

19. Clerk A and Sugden PH. Small guanine nucleotide-binding proteins and myocardial hypertrophy. Circ Res 86: 1019-1023, 2000.[Abstract/Free Full Text]

20. Colavitti R, Pani G, Bedogni B, Anzevino R, Borrello S, Waltenberger J, and Galeotti T. Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR. J Biol Chem 277: 3101-3108, 2002.[Abstract/Free Full Text]

21. Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, and Gutkind JS. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 81: 1137-1146, 1995.[ISI][Medline]

22. Dang PM, Cross AR, and Babior BM. Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67PHOX and cytochrome b558. Proc Natl Acad Sci USA 98: 3001-3005, 2001.[Abstract/Free Full Text]

23. Debant A, Serra-Pages C, Seipel K, O'Brien S, Tang M, Park SH, and Streuli M. The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rhospecific guanine nucleotide exchange factor domains. Proc Natl Acad Sci USA 93: 5466-5471, 1996.[Abstract/Free Full Text]

24. Del Pozo MA, Kiosses WB, Alderson NB, Meller N, Hahn KM, and Schwartz MA. Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nat Cell Biol 4: 232-239, 2002.[ISI][Medline]

25. Diekmann D, Abo A, Johnston C, Segal AW, and Hall A. Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity. Science 265: 531-533, 1994.[ISI][Medline]

26. Doanes AM, Irani K, Goldschmidt-Clermont PJ, and Finkel T. A requirement for rac1 in the PDGF-stimulated migration of fibroblasts and vascular smooth cells. Biochem Mol Biol Int 45: 279-287, 1998.[ISI][Medline]

27. Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, and Weinberg RA. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature 363: 45-51, 1993.[ISI][Medline]

28. Faruqi TR, Gomez D, Bustelo XR, Bar-Sagi D, and Reich NC. Rac1 mediates STAT3 activation by autocrine IL-6. Proc Natl Acad Sci USA 98: 9014-9019, 2001.[Abstract/Free Full Text]

29. Fukui T, Ishizaka N, Rajagopalan S, Laursen JB, Capers Q, Taylor WR, Harrison DG, de Leon H, Wilcox JN, and Griendling KK. p22phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats. Circ Res 80: 45-51, 1997.[Abstract/Free Full Text]

30. Goldschmidt-Clermont PJ and Moldovan L. Stress, superoxide, and signal transduction. Gene Expr 7: 255-260, 1999.[ISI][Medline]

31. Griendling KK, Minieri CA, Ollerenshaw JD, and Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 1141-1148, 1994.[Abstract]

32. Griendling KK, Sorescu D, and Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 86: 494-501, 2000.[Abstract/Free Full Text]

33. Han J, Luby-Phelps K, Das B, Shu X, Xia Y, Mosteller RD, Krishna UM, Falck JR, White MA, and Broek D. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 279: 558-560, 1998.[Abstract/Free Full Text]

34. Hartwig JH. Mechanisms of actin rearrangements mediating platelet activation. J Cell Biol 118: 1421-1442, 1992.[Abstract]

35. Hartwig JH, Bokoch GM, Carpenter CL, Janmey PA, Taylor LA, Toker A, and Stossel TP. Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell 82: 643-653, 1995.[ISI][Medline]

36. He Q and LaPointe MC. Src and Rac mediate endothelin-1 and lysophosphatidic acid stimulation of the human brain natriuretic peptide promoter. Hypertension 37: 478-484, 2001.[Abstract/Free Full Text]

37. Heitzer T, Schlinzig T, Krohn K, Meinertz T, and Munzel T. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation 104: 2673-2678, 2001.[Abstract/Free Full Text]

38. Hooshmand-Rad R, Claesson-Welsh L, Wennstrom S, Yokote K, Siegbahn A, and Heldin CH. Involvement of phosphatidylinositide 3'-kinase and Rac in platelet-derived growth factor-induced actin reorganization and chemotaxis. Exp Cell Res 234: 434-441, 1997.[ISI][Medline]

39. Innocenti M, Tenca P, Frittoli E, Faretta M, Tocchetti A, Di Fiore PP, and Scita G. Mechanisms through which Sos-1 coordinates the activation of Ras and Rac. J Cell Biol 156: 125-136, 2002.[Abstract/Free Full Text]

40. Irani K. Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ Res 87: 179-183, 2000.[Abstract/Free Full Text]

41. Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, Sundaresan M, Finkel T, and Goldschmidt-Clermont PJ. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 275: 1649-1652, 1997.[Abstract/Free Full Text]

42. Jones GE, Allen WE, and Ridley AJ. The Rho GTPases in macrophage motility and chemotaxis. Cell Adhes Commun 6: 237-245, 1998.[ISI][Medline]

43. Katzav S, Cleveland JL, Heslop HE, and Pulido D. Loss of the amino-terminal helix-loop-helix domain of the vav protooncogene activates its transforming potential. Mol Cell Biol 11: 1912-1920, 1991.[ISI][Medline]

44. Kiosses WB, Shattil SJ, Pampori N, and Schwartz MA. Rac recruits high-affinity integrin {alpha}v{beta}3 to lamellipodia in endothelial cell migration. Nat Cell Biol 3: 316-320, 2001.[ISI][Medline]

45. Knaus UG, Heyworth PG, Evans T, Curnutte JT, and Bokoch GM. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science 254: 1512-1515, 1991.[ISI][Medline]

46. Kozma R, Ahmed S, Best A, and Lim L. The GTPase-activating protein n-chimaerin cooperates with Rac1 and Cdc42Hs to induce the formation of lamellipodia and filopodia. Mol Cell Biol 16: 5069-5080, 1996.[Abstract]

47. Kraynov VS, Chamberlain C, Bokoch GM, Schwartz MA, Slabaugh S, and Hahn KM. Localized Rac activation dynamics visualized in living cells. Science 290: 333-337, 2000.[Abstract/Free Full Text]

48. Kutsche K, Yntema H, Brandt A, Jantke I, Nothwang HG, Orth U, Boavida MG, David D, Chelly J, Fryns JP, Moraine C, Ropers HH, Hamel BC, van Bokhoven H, and Gal A. Mutations in ARHGEF6, encoding a guanine nucleotide exchange factor for Rho GTPases, in patients with X-linked mental retardation. Nat Genet 26: 247-250, 2000.[ISI][Medline]

49. Lander HM. An essential role for free radicals and derived species in signal transduction. FASEB J 11: 118-124, 1997.[Abstract/Free Full Text]

50. Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM, and Harrison DG. Role of p47phox in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 40: 511-515, 2002.[Abstract/Free Full Text]

51. Laufs U, Kilter H, Konkol C, Wassmann S, Bohm M, and Nickenig G. Impact of HMG CoA reductase inhibition on small GTPases in the heart. Cardiovasc Res 53: 911-920, 2002.[ISI][Medline]

52. Lee ZW, Kweon SM, Kim SJ, Kim JH, Cheong C, Park YM, and Ha KS. The essential role of H2O2 in the regulation of intracellular Ca2+ by epidermal growth factor in rat-2 fibroblasts. Cell Signal 12: 91-98, 2000.[ISI][Medline]

53. Li C, Hu Y, Mayr M, and Xu Q. Cyclic strain stress-induced mitogen-activated protein kinase (MAPK) phosphatase 1 expression in vascular smooth muscle cells is regulated by Ras/Rac-MAPK pathways. J Biol Chem 274: 25273-25280, 1999.[Abstract/Free Full Text]

54. Li C, Hu Y, Sturm G, Wick G, and Xu Q. Ras/Rac-dependent activation of p38 mitogen-activated protein kinases in smooth muscle cells stimulated by cyclic strain stress. Arterioscler Thromb Vasc Biol 20: E1-E9, 2000.[Medline]

55. Li JM and Shah AM. Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells. J Biol Chem 277: 19952-19960, 2002.[Abstract/Free Full Text]

56. Lopes NH, Vasudevan SS, Gregg D, Selvakumar B, Pagano PJ, Kovacic H, and Goldschmidt-Clermont PJ. Rac-dependent monocyte chemoattractant protein-1 production is induced by nutrient deprivation. Circ Res 91: 798-805, 2002.[Abstract/Free Full Text]

57. Massy ZA, Keane WF, and Kasiske BL. Inhibition of the mevalonate pathway: benefits beyond cholesterol reduction? Lancet 347: 102-103, 1996.[ISI][Medline]

58. Mayr M, Hu Y, Hainaut H, and Xu Q. Mechanical stress-induced DNA damage and rac-p38MAPK signal pathways mediate p53-dependent apoptosis in vascular smooth muscle cells. FASEB J 16: 1423-1425, 2002.[Abstract/Free Full Text]

59. Minden A, Lin A, Claret FX, Abo A, and Karin M. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 81: 1147-1157, 1995.[ISI][Medline]

60. Moldovan L, Moldovan NI, Sohn RH, Parikh SA, and Goldschmidt-Clermont PJ. Redox changes of cultured endothelial cells and actin dynamics. Circ Res 86: 549-557, 2000.[Abstract/Free Full Text]

61. Nishiyama T, Sasaki T, Takaishi K, Kato M, Yaku H, Araki K, Matsuura Y, and Takai Y. rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells. Mol Cell Biol 14: 2447-2456, 1994.[Abstract]

62. Nowicki PT, Flavahan S, Hassanain H, Mitra S, Holland S, Goldschmidt-Clermont PJ, and Flavahan NA. Redox signaling of the arteriolar myogenic response. Circ Res 89: 114-116, 2001.[Abstract/Free Full Text]

63. Orrico A, Galli L, Falciani M, Bracci M, Cavaliere ML, Rinaldi MM, Musacchio A, and Sorrentino V. A mutation in the pleckstrin homology (PH) domain of the FGD1 gene in an Italian family with faciogenital dysplasia (Aarskog-Scott syndrome). FEBS Lett 478: 216-220, 2000.[ISI][Medline]

64. Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C, Ballinger CA, Brasier AR, Bode C, and Runge MS. Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47phox may participate in forming this oxidase in vitro and in vivo. J Biol Chem 274: 19814-19822, 1999.[Abstract/Free Full Text]

65. Pimentel DR, Amin JK, Xiao L, Miller T, Viereck J, Oliver-Krasinski J, Baliga R, Wang J, Siwik DA, Singh K, Pagano P, Colucci WS, and Sawyer DB. Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes. Circ Res 89: 453-460, 2001.[Abstract/Free Full Text]

66. Pracyk JB, Hegland DD, and Tanaka K. Effect of a dominant negative ras on myocardial hypertrophy by using adenoviral-mediated gene transfer. Surgery 122: 404-410, 1997.[ISI][Medline]

67. Pracyk JB, Tanaka K, Hegland DD, Kim KS, Sethi R, Rovira II, Blazina DR, Lee L, Bruder JT, Kovesdi I, Goldshmidt-Clermont PJ, Irani K, and Finkel T. A requirement for the rac1 GTPase in the signal transduction pathway leading to cardiac myocyte hypertrophy. J Clin Invest 102: 929-937, 1998.[Abstract/Free Full Text]

68. Price MO, Atkinson SJ, Knaus UG, and Dinauer MC. Rac activation induces NADPH oxidase activity in transgenic COSphox cells and level of superoxide production is exchange factor-dependent. J Biol Chem 2002.

69. Qiu RG, Chen J, Kirn D, McCormick F, and Symons M. An essential role for Rac in Ras transformation. Nature 374: 457-459, 1995.[ISI][Medline]

70. Quilliam LA, Huff SY, Rabun KM, Wei W, Park W, Broek D, and Der CJ. Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity. Proc Natl Acad Sci USA 91: 8512-8516, 1994.[Abstract]

71. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, and Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest 97: 1916-1923, 1996.[Abstract/Free Full Text]

72. Rey FE, Cifuentes ME, Kiarash A, Quinn MT, and Pagano PJ. Novel competitive inhibitor of NAD(P)H oxidase assembly attenuates vascular O2- and systolic blood pressure in mice. Circ Res 89: 408-414, 2001.[Abstract/Free Full Text]

73. Ridley AJ. Rho family proteins: coordinating cell responses. Trends Cell Biol 11: 471-477, 2001.[ISI][Medline]

74. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, and Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70: 401-410, 1992.[ISI][Medline]

75. Roberts AW, Kim C, Zhen L, Lowe JB, Kapur R, Petryniak B, Spaetti A, Pollock JD, Borneo JB, Bradford GB, Atkinson SJ, Dinauer MC, and Williams DA. Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity 10: 183-196, 1999.[ISI][Medline]

76. Sanchez-Madrid F, and Del Pozo MA. Leukocyte polarization in cell migration and immune interactions. EMBO J 18: 501-511, 1999.[Abstract/Free Full Text]

77. Schmitz U, Thommes K, Beier I, Wagner W, Sachinidis A, Dusing R, and Vetter H. Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck. J Biol Chem 276: 22003-22010, 2001.[Abstract/Free Full Text]

78. Scita G, Nordstrom J, Carbone R, Tenca P, Giardina G, Gutkind S, Bjarnegard M, Betsholtz C, and Di Fiore PP. EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401: 290-293, 1999.[ISI][Medline]

79. Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M, and Di Fiore PP. An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine. J Cell Biol 154: 1031-1044, 2001.[Abstract/Free Full Text]

80. Scita G, Tenca P, Frittoli E, Tocchetti A, Innocenti M, Giardina G, and Di Fiore PP. Signaling from Ras to Rac and beyond: not just a matter of GEFs. EMBO J 19: 2393-2398, 2000.[Abstract/Free Full Text]

81. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, and Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res 91: 406-413, 2002.[Abstract/Free Full Text]

82. Settleman J, Narasimhan V, Foster LC, and Weinberg RA. Molecular cloning of cDNAs encoding the GAP-associated protein p190: implications for a signaling pathway from ras to the nucleus. Cell 69: 539-549, 1992.[ISI][Medline]

83. Simon MA, Dodson GS, and Rubin GM. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell 73: 169-177, 1993.[ISI][Medline]

84. Soga N, Connolly JO, Chellaiah M, Kawamura J, and Hruska KA. Rac regulates vascular endothelial growth factor stimulated motility. Cell Adhes Commun 8: 1-13, 2001.

85. Soulet C, Gendreau S, Missy K, Benard V, Plantavid M, and Payrastre B. Characterisation of Rac activation in thrombin- and collagen-stimulated human blood platelets. FEBS Lett 507: 253-258, 2001.[ISI][Medline]

86. Sundaresan M, Yu ZX, Ferrans VJ, Sulciner DJ, Gutkind JS, Irani K, Goldschmidt-Clermont PJ, and Finkel T. Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem J 318: 379-382, 1996.[ISI][Medline]

87. Sussman MA, Welch S, Walker A, Klevitsky R, Hewett TE, Price RL, Schaefer E, and Yager K. Altered focal adhesion regulation correlates with cardiomyopathy in mice expressing constitutively active rac1. J Clin Invest 105: 875-886, 2000.[Abstract/Free Full Text]

88. Suzuki-Inoue K, Yatomi Y, Asazuma N, Kainoh M, Tanaka T, Satoh K, and Ozaki Y. Rac, a small guanosine triphosphate-binding protein, and p21-activated kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin {alpha}2{beta}1. Blood 98: 3708-3716, 2001.[Abstract/Free Full Text]

89. Szocs K, Lassegue B, Sorescu D, Hilenski LL, Valppu L, Couse TL, Wilcox JN, Quinn MT, Lambeth JD, and Griendling KK. Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury. Arterioscler Thromb Vasc Biol 22: 21-27, 2002.[Abstract/Free Full Text]

90. Takai Y, Sasaki T, and Matozaki T. Small GTP-binding proteins. Physiol Rev 81: 153-208, 2001.[Abstract/Free Full Text]

91. Takemoto M and Liao JK. Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors. Arterioscler Thromb Vasc Biol 21: 1712-1719, 2001.[Abstract/Free Full Text]

92. Takemoto M, Node K, Nakagami H, Liao Y, Grimm M, Takemoto Y, Kitakaze M, and Liao JK. Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. J Clin Invest 108: 1429-1437, 2001.[Abstract/Free Full Text]

93. Thakur NK, Hayashi T, Sumi D, Kano H, Tsunekawa T, and Iguchi A. HMG-CoA reductase inhibitor stabilizes rabbit atheroma by increasing basal NO and decreasing superoxide. Am J Physiol Heart Circ Physiol 281: H75-H83, 2001.[Abstract/Free Full Text]

94. Touyz RM and Schiffrin EL. Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells. Pharmacol Rev 52: 639-672, 2000.[Abstract/Free Full Text]

95. Ushio-Fukai M, Alexander RW, Akers M, and Griendling KK. p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy. J Biol Chem 273: 15022-15029, 1998.[Abstract/Free Full Text]

96. Ushio-Fukai M, Griendling KK, Becker PL, Hilenski L, Halleran S, and Alexander RW. Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 21: 489-495, 2001.[Abstract/Free Full Text]

97. Ushio-Fukai M, Zafari AM, Fukui T, Ishizaka N, and Griendling KK. p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem 271: 23317-23321, 1996.[Abstract/Free Full Text]

98. Van Aelst L and D'Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev 11: 2295-2322, 1997.[Free Full Text]

99. Van Wetering S, van Buul JD, Quik S, Mul FP, Anthony EC, ten Klooster JP, Collard JG, and Hordijk PL. Reactive oxygen species mediate Rac-induced loss of cell-cell adhesion in primary human endothelial cells. J Cell Sci 115: 1837-1846, 2002.[Abstract/Free Full Text]

100. Vecchione C and Brandes RP. Withdrawal of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors elicits oxidative stress and induces endothelial dysfunction in mice. Circ Res 91: 173-179, 2002.[Abstract/Free Full Text]

101. Vouret-Craviari V, Boquet P, Pouyssegur J, and Obberghen-Schilling E. Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function. Mol Biol Cell 9: 2639-2653, 1998.[Abstract/Free Full Text]

102. Wang HD, Xu S, Johns DG, Du Y, Quinn MT, Cayatte AJ, and Cohen RA. Role of NADPH oxidase in the vascular hypertrophic and oxidative stress response to angiotensin II in mice. Circ Res 88: 947-953, 2001.[Abstract/Free Full Text]

103. Warnholtz A, Nickenig G, Schulz E, Macharzina R, Brasen JH, Skatchkov M, Heitzer T, Stasch JP, Griendling KK, Harrison DG, Bohm M, Meinertz T, and Munzel T. Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the reninangiotensin system. Circulation 99: 2027-2033, 1999.[Abstract/Free Full Text]

104. Wassmann S, Laufs U, Baumer AT, Muller K, Ahlbory K, Linz W, Itter G, Rosen R, Bohm M, and Nickenig G. HMG-CoA reductase inhibitors improve endothelial dysfunction in normocholesterolemic hypertension via reduced production of reactive oxygen species. Hypertension 37: 1450-1457, 2001.[Abstract/Free Full Text]

105. Wassmann S, Laufs U, Baumer AT, Muller K, Konkol C, Sauer H, Bohm M, and Nickenig G. Inhibition of geranylgeranylation reduces angiotensin II-mediated free radical production in vascular smooth muscle cells: involvement of angiotensin AT1 receptor expression and Rac1 GTPase. Mol Pharmacol 59: 646-654, 2001.[Abstract/Free Full Text]

106. Westwick JK, Lambert QT, Clark GJ, Symons M, Van Aelst L, Pestell RG, and Der CJ. Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways. Mol Cell Biol 17: 1324-1335, 1997.[Abstract]

107. Wojciak-Stothard B, Entwistle A, Garg R, and Ridley AJ. Regulation of TNF-alpha-induced reorganization of the actin cytoskeleton and cell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells. J Cell Physiol 176: 150-165, 1998.[ISI][Medline]

108. Woo CH, You HJ, Cho SH, Eom YW, Chun JS, Yoo YJ, and Kim JH. Leukotriene B4 stimulates Rac-ERK cascade to generate reactive oxygen species that mediates chemotaxis. J Biol Chem 277: 8572-8578, 2002.[Abstract/Free Full Text]

109. Wung BS, Cheng JJ, Shyue SK, and Wang DL. NO modulates monocyte chemotactic protein-1 expression in endothelial cells under cyclic strain. Arterioscler Thromb Vasc Biol 21: 1941-1947, 2001.[Abstract/Free Full Text]

110. Xu Q, Schett G, Li C, Hu Y, and Wick G. Mechanical stress-induced heat shock protein 70 expression in vascular smooth muscle cells is regulated by Rac and Ras small G proteins but not mitogen-activated protein kinases. Circ Res 86: 1122-1128, 2000.[Abstract/Free Full Text]

111. Yeh LH, Park YJ, Hansalia RJ, Ahmed IS, Deshpande SS, Goldschmidt-Clermont PJ, Irani K, and Alevriadou BR. Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS. Am J Physiol Cell Physiol 276: C838-C847, 1999.[Abstract/Free Full Text]

112. Yusuf S, Dagenais G, Pogue J, Bosch J, and Sleight P. Vitamin E supplementation and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med 342: 154-160, 2000.[Abstract/Free Full Text]

113. Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, and Griendling KK. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy. Hypertension 32: 488-495, 1998.[Abstract/Free Full Text]

114. Zalba G, San Jose G, Moreno MU, Fortuno MA, Fortuno A, Beaumont FJ, and Diez J. Oxidative stress in arterial hypertension: role of NAD(P)H oxidase. Hypertension 38: 1395-1399, 2001.[Abstract/Free Full Text]

115. Zhou K, Wang Y, Gorski JL, Nomura N, Collard J, and Bokoch GM. Guanine nucleotide exchange factors regulate specificity of downstream signaling from Rac and Cdc42. J Biol Chem 273: 16782-16786, 1998.[Abstract/Free Full Text]