Emerging Role of Oxidative Stress in Metabolic Syndrome and

Cardiovascular Diseases: Important Role of Rac/NADPH Oxidase

Mohammad T. Elnakish1,2, Hamdy H. Hassanain1, Paul M.L Janssen1,2, Mark G. Angelos1,3 and

Mahmood Khan*1,3

1Dorothy M. Davis Heart and Lung Research Institute,

2Department of Physiology and Cell Biology, 3Department of Emergency Medicine,

The Ohio State University Wexner Medical Center, Columbus, OH, USA

Key Words: Oxidative Stress, Metabolic syndrome, Cardiovascular disease

*Address for Correspondence:

Mahmood Khan, M. Pharm, Ph.D.

Department of Emergency Medicine

The Ohio State University Wexner Medical Center

420 W. 12th Ave, Room 110

Columbus, OH 43210, USA

Tel: 614-688-7803

E-mail: mahmood.khan@osumc.edu

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/path.4255

Abstract

Oxidative stress is a term defining states of elevated reactive oxygen species (ROS)

levels. Normally, ROS control several physiological processes such as host defense, biosynthesis

of hormones, fertilization, and cellular signaling. However, oxidative stress has been involved in

different pathologies including metabolic syndrome and numerous cardiovascular diseases. A

major source of ROS involved in both metabolic syndrome and cardiovascular pathophysiology

is the NADPH oxidase (NOX) family of enzymes. NOX is a multi-component enzyme complex

that consists of membrane-bound cytochrome b-558, which is a heterodimer of gp91phox and

p22phox, cytosolic regulatory subunits p47phox and p67phox, and the small GTP-binding

protein Rac1. Rac1 plays many important biological functions in cells, but perhaps the most

unique function of Rac1 is its ability to bind and activate the NOX complex. Furthermore, Rac1

has been reported to be a key regulator of oxidative stress through its co-regulatory effects on

both nitric oxide (NO) synthase and NOX. Therefore, the main goal of this review is to give a

brief outline about the important role of Rac1/NOX axis in the pathophysiology of both

metabolic syndrome and cardiovascular disease.

Key Words: Oxidative stress, Metabolic syndrome, Cardiovascular disease

Introduction

Oxidative stress is a term defining states characterized by elevated reactive oxygen

species (ROS) levels. ROS are reactive chemical units involving two main categories; 1) free

radicals such as superoxide (O2.-), hydroxyl (OH.) and nitric oxide (NO.), and 2) non-radical

derivatives of O2 such as hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) [1]. Normally,

ROS control several physiological processes such as host defense, biosynthesis of hormones,

fertilization, and cellular signaling. However, oxidative stress is also involved in different

pathologies including metabolic syndrome and numerous cardiovascular diseases [1, 2].

Generally, oxidative stress results in damage to proteins, lipids and DNA, resulting in cellular

dysfunction [3]. In particular, an excess of O2.- may decrease nitric oxide (NO) availability,

resulting in endothelial dysfunction and a decrease in endothelium-dependent vasodilation [4].

Additionally, oxidative modification of proteins may result in the formation of nitrotyrosine,

which represents a powerful and autonomous marker of cardiovascular diseases [5].

Furthermore, O2.- is implicated in the generation of oxidized LDL (oxLDL), a key initiator of

atherosclerosis [6].

A major source of ROS involved in both metabolic syndrome and cardiovascular

pathophysiology is the NADPH oxidase (NOX) family of enzymes [2, 7]. NOX is a multi-

component enzyme complex that consists of membrane-bound cytochrome b-558 (which is a

heterodimer of gp91phox and p22phox), cytosolic regulatory subunits (p47phox and p67phox)

and the small GTP-binding protein Rac1 [8]. Rac1 performs many important biological functions

in cells, but perhaps the most unique is its ability to bind and activate the NOX complex [9].

Upon enzyme activation, translocation as well as assembly of the cytosolic subunits to the

membrane subunits takes place. In the activated enzyme complex, the catalytic subunit acts as an

electron transport system that utilizes NADPH as an electron donor to transfer electrons to

molecular oxygen, leading to the formation of O2.- [10, 11]. Furthermore, Rac1 has been reported

to be a key regulator of oxidative stress through its co-regulatory effects on both NO synthase

and NOX [12].

Role of Rac1/NADPH Oxidase in Metabolic Syndrome and Associated Risk Factors

The metabolic syndrome is an assemblage of insulin resistance and hyperinsulinemia,

impaired glucose tolerance or diabetes, dyslipidemia, obesity, and hypertension. Metabolic

syndrome has reached epidemic levels in developed countries, where its occurrence might

exceed 40% of subjects > 40 years old. A frequent association has been identified between

metabolic syndrome and cardiovascular diseases, particularly atherosclerosis, which is

responsible for a significant portion of the morbidity and mortality associated with metabolic

syndrome [13-15].

Existing evidence supports the correlation of metabolic syndrome with increased

systemic oxidative stress. For example, Hansel et al [16] have shown that metabolic syndrome is

associated with impaired antioxidative activity of high density lipoprotein (HDL) sub-fractions,

elevated systemic oxidative stress and insulin resistance. Additionally, Holvoet et al [17]

reported that metabolic syndrome is coupled with levels of circulating oxLDL that are linked to a

larger disposition to atherothrombotic coronary disease. Interestingly, phagocytic NOX–

dependent O2.- production is considerably increased in patients with metabolic syndrome.

Evidence of this increased oxidative stress is manifest by increased oxLDL and nitrotyrosine

levels as well as subclinical atherosclerosis related to the NOX activity [2]. Oxidative stress has

also been shown to play a key role in the pathogenesis of primary risk factors associated with

metabolic syndrome such as obesity, diabetes and hypertension (Figure 1).

Rac1/NADPH Oxidase in Obesity

Oxidative stress in accumulated fat has been found to be a key pathogenic mechanism of

obesity-associated metabolic syndrome. Fat accumulation correlates with systemic oxidative

stress both in humans and mice. Notably, ROS production shows a selective increase in adipose

tissue of obese mice, followed by enhanced expression of NOX and suppressed antioxidative

enzyme expression [18]. Aside from higher oxidative stress states, increased NOX activity and

Rac1 up-regulation have been detected in overweight Chinese adolescents. In this population, a

significant positive bivariate correlation was found between Rac1 expression, increased

oxidative stress and obesity-related indices, signifying that Rac1 may act as a link between

obesity and oxidative stress in obese subjects [3].

Rac1/ NADPH Oxidase in Diabetes

Increased ROS generation is considered essential to the development of diabetes. Under

diabetic conditions, oxidative stress decreases glucose uptake in muscle and fat and impairs

insulin release from pancreatic β cells [19-21]. Several reports described Rac1/NOX as a key

determinant in regulating glucose/insulin-dependent metabolic processes with the possibility of

being a main contributor in the pathogenesis of diabetes. Rac1 is also a positive modulator in

regulating glucose-stimulated insulin secretion via inducing cytoskeletal remodeling to

encourage granule mobilization to the plasma membrane for fusion and insulin release [22],

Rac1-mediated NOX activation and ROS production [23, 24]. Additionally, Rac1 activation

increases insulin-stimulated glucose uptake into skeletal muscle, a crucial step for glucose

homoeostasis [25]. On the other hand, Rac1 plays a negative modulatory role in islet function by

promoting NOX-mediated ROS production; this leads to induction of oxidative stress and

metabolic dysregulation of the islet β-cell under the effects of glucolipotoxicity, cytokines, and

ceramide [25-27]. In diabetic human islets as well as in islets from a well-known model of

obesity and type 2 diabetes, ZDF, accelerated Rac1/NOX/ROS signaling may be responsible for

the onset of mitochondrial dysregulation in diabetes [28]. Recently, augmented NOX activity in

monocytes was also reported to underlie oxidative stress of patients with type 2 diabetes [29].

Numerous studies have revealed that ROS is a key factor in the development of

cardiovascular complications, which represent the primary cause of death in diabetic patients

[30]. Shen et al [31] have shown that Rac1, through NOX activation, promotes mitochondrial

ROS generation and plays an important role in cardiomyocyte apoptosis and myocardial

dysfunction in a diabetic mouse model. Likewise, the same group has shown that Rac1, through

NOX oxidase activation, induces myocardial remodeling and dysfunction in a mouse model of

type 1 diabetes. The effects have been related to endoplasmic reticulum stress and inflammation

[32]. Additionally, Rac1 plays a vital role in murine experimental diabetes-induced vascular

injury [33]. Furthermore, it has been exhibited that disruption of Rac1 and inhibition of calpain

attenuate myocardial injury and improve functional recovery in diabetic hearts after ischemia-

reperfusion (I/R). In this model, Rac1 signaling is partially mediated via NOX-stimulated ROS

generation and subsequent calpain activation [34]. Moreover, NOX-stimulated ROS is involved

in impaired post-ischemic neovascularization, which could be responsible for the poor outcome

after vascular occlusion in diabetes [30]. Overall, the Rac1/NOX pathway has been proposed as a

potentially valuable therapeutic target for the treatment of diabetic cardiovascular complications.

Rac1/ NADPH Oxidase in Hypertension

Increasing evidence indicates that ROS play an important role in the pathophysiology of

hypertension. Oxidative stress is implicated in a variety of hypertensive disorders, including

lead-induced hypertension, uremic hypertension, cyclosporine-induced hypertension, salt-

sensitive hypertension, pre-eclampsia, and essential hypertension [35, 36]. Particularly,

angiotensin II-induced hypertension has been associated with elevated vascular NOX-mediated

O2.- levels [37]. Interestingly, Rac1 is positioned downstream in the angiotensin II pathway,

where it directly regulates NOX activation by inducing rapid translocation of Rac1-GTPase to

the cell membrane [38]. We have shown that oxidant signaling by the Rac1/NOX/ROS pathway

is essential for the myogenic response of arterioles; a mechanism that may contribute to

heightened constriction and oxidant stress in hypertension [39]. Additionally, we demonstrated

that over-expression of the human cDNA of the constitutively active mutant of Rac1 (Rac-CA)

in vascular smooth muscle cells (VSMCs) resulted in excessive amounts of NOX-mediated O2.-

in the vessel wall that led to heightened production of ONOO- and reduced NO levels, and

development of hypertension in the Rac-CA transgenic mice [40]. Similarly, activation of Rac1-

mediated NOX promotes O2.- production in the nucleus tractus solitaires of stroke-prone

spontaneously hypertensive rats (SPSHRs), and this mechanism might be important for the

neuropathogenesis of hypertension in these rats [41]. Recently, abnormal induction of the

aldosterone/mineralocorticoid receptor pathway has been implicated in the development of salt-

induced hypertension and cardiovascular damage in metabolic syndrome [42, 43]. The same

group identified active Rac1 as a novel modulator of mineralocorticoid receptor pathway, salt

sensitivity and salt-induced hypertension and renal injury. They also reported that both salt and

obesity activate Rac1 and cause activation of the mineralocorticoid receptor pathway, which

plays a key role in the development of salt-sensitive hypertension and renal injury in metabolic

syndrome [44] (Figure 2).

Role of Rac1/ NADPH Oxidase in Different cardiovascular diseases

A plethora of data confirms the role of oxidative stress in the pathogenesis of different

cardiovascular diseases including vascular injury, atherosclerosis, cardiac remodeling, heart

failure, arrhythmias and ischemia-reperfusion injury. Furthermore, a pathway-based genome-

wide association analysis has recognized Rac1 as one of the biologically central genes in

coronary heart disease [45].

Rac1/ NADPH Oxidase in Vascular Remodeling and Atherosclerosis

Several studies have recognized the role of ROS in growth processes associated with

vascular injury and remodeling. For instance, Angiotensin II-induced hypertrophy or hyperplasia

of VSMCs has been linked to NOX-mediated O2.- generation [49-51]. The enhanced NOX-

stimulated O2.- is involved in developed vascular wall hypertrophy of the SHRs [46]. Also, over-

expression of the p22phox subunit in VSMCs results in increased ROS and vascular hypertrophy

in transgenic mice [47]. These results from experimental animals have been confirmed in human

studies showing that increased NOX-mediated ROS plays a role in the angiotensin II-induced

vascular remodeling in essential hypertension [48]. In addition, biomechanical stress induced by

high blood pressure triggers integrin-linked kinase 1/βPIX/Rac1 signaling, which results in

vascular dysfunction by activating NOX-mediated O2.- generation [49]. In line with these

findings, we have observed a key role for vascular Rac1 in regulating and maintaining normal

vascular tone of the blood vessels, blood pressure and subsequent changes in vascular structure

of resistance arteries. Similar to essential hypertension, hypertensive Rac-CA transgenic mice

exhibited significant increases in the tail artery O2.- levels, which is associated with inward

eutrophic remodeling (reduced lumen diameter, increased ratio of “media thickness / lumen

diameter” and unchanged cross-sectional area of the media). These changes are attenuated in the

tail arteries of transgenic mice over-expressing the dominant negative isoform (Rac-DN) [Coats

P & Hassanain HH, unpublished]. Rac-dependent activation of the nuclear factor-κB (NF-κB)

and ROS are also associated with enhanced thrombogenicity of the vessel wall contributing to

vascular remodeling in pulmonary hypertension [50] (Figure 2).

On the other hand, ROS seem to be implicated in the pathogenesis of atherosclerosis via a

sequence of molecular changes that finally results in macrophage infiltration of the endothelium

and plaque formation. In the atherosclerotic process, ROS can promote oxLDL formation,

stimulate matrix metalloproteinases (MMPs), improve VSMC growth and provoke

inflammatory mediator production, including MMP-1, intercellular adhesion molecule 1 (ICAM-

1) and vascular cellular adhesion molecule 1 (VCAM-1). ROS can also cause arterial

dysfunction by inhibiting NO, a potent vasodilator and anti-aggregating molecule released by

endothelium [51] (Figure 2). Vendrov et al [52] showed that absence of functional NOX in

P47phox-deficient animals suppressed atherosclerotic lesion growth by restricting recruitment of

the macrophages to the endothelial surface and by modulating expression of adhesive molecules,

such as ICAM-1 and VCAM-1. In this way NOX activation may contribute to the

initiation/progression of the atherosclerotic lesion. Additionally, Warnholtz et al [53] reported

that hypercholesterolemia is coupled with up-regulation of angiotensin receptors, endothelial

dysfunction, and enhanced NOX-mediated vascular O2.- generation. They also showed that

angiotensin receptor blockers improved endothelial dysfunction, inhibited NOX-mediated

vascular O2.-, and reduced early plaque formation, suggesting a crucial role of angiotensin II-

mediated O2.- generation in the early stage of atherosclerosis. A role for vascular Rac1/NOX-

mediated ROS in the development of atherosclerosis in an atherogenic mouse model has also

been described [54]. Likewise, physical inactivity increases vascular Rac1/NOX-mediated ROS,

which contributes to endothelial dysfunction and atherosclerosis as opposed to physically active

lifestyle [55]. Furthermore, a study by Ohkawara et al [56] showed evidence of significant

increases in RhoA and Rac1 with activity in the progression of atherosclerosis in Watanabe

heritable hyperlipidemic rabbits. Moreover, Rac1-mediated signaling plays a central role in

secretion-dependent platelet aggregation in blood activated by a wide range of platelet agonists

including atherosclerotic plaque [57]. These findings were further confirmed in humans. Azumi

et al [58] demonstrated that p22phox, the membrane-bound subunit of NOX, was over-expressed

in the vessel wall of atherosclerotic coronary arteries compared to non-atherosclerotic vessels.

Similarly, in patients undergoing heart transplantation, Guzik et al [59] found that O2.- was over-

expressed in atherosclerotic coronary arteries compared with non-atherosclerotic arteries and that

NOX predominantly contributed to this increase. Consistent with these results, several NOX

proteins, such as gp91phox and NOX4, were involved in cell-specific increased intracellular

oxidative stress in human coronary atherosclerosis and consequently may contribute to the

genesis and progression of human coronary atherosclerotic disease [60].

Rac1/ NADPH Oxidase in Cardiac Remodeling and Heart Failure

Various studies indicate that many harmful cellular phenotypes detected in hypertrophied

and failing myocardium are accredited to ROS and oxidative stress [61]. The therapeutic

applications of antioxidants to experimental cardiac hypertrophy have shown a decline in cardiac

mass and maintenance of cardiac function, signifying an important role of ROS in cardiac

structural remodeling [62, 63]. Also, β-adrenoreceptor stimulation provokes cardiac oxidative

stress that may be chronically responsible for the development of cardiac remodeling [64].

Recently, NOX through redox-sensitive signal transduction has been presented as a key player in

the pathogenesis of several aspects of cardiac remodeling and its antecedent conditions.

Additionally, Rac1-GTPase activation was found to play a main role in the initiation of cardiac

hypertrophy linking NOX related ROS production to the hypertrophic signaling cascade [8]. In

support of the role of NOX in cardiac hypertrophy, mice with targeted disruption of gp91phox

have decreased myocardial O2.-production and develop less cardiac hypertrophy in response to

angiotensin II [65]. Likewise, Satoh et al [66] showed that Rac1 induces NOX activity and is the

main regulator of cardiac hypertrophy in response to angiotensin II in adult mouse

cardiomyocytes. Rac1-mediated O2.- generation also plays a crucial role in the development of

both angiotensin II- and transaortic constriction-induced cardiac hypertrophy [67-69]. In

conjunction with these studies, our laboratory [70] and others [7] have shown that Rac1/NOX-

mediated O2.- is involved in cardiomyocyte hypertrophy in aging rodents. Recently, our

laboratory has shown that Rac1 activation is partially involved in thyroxin (T4)-induced

cardiomyocyte hypertrophy [71]. Also, Lezoualc’h et al [72] showed the importance of

cardiomyocyte Rac1 in the development of cardiac hypertrophy in transgenic mice. In an elegant

study, Sussman et al. [73] reported a transgenic mouse model that over-expresses human Rac1

cDNA in the heart using the α-myosin heavy chain promoter. The characterization of human

Rac1-expressing transgenic mice (RacET mice) revealed an unusual dichotomy: 1) rapid-onset,

high-level postnatal expression that led to lethal dilated cardiomyopathy and 2) slow-onset

postnatal expression leading to transient hypertrophy with survival that was evident in 3-week-

old RacET ventricles and resolved with age. Promoter induction in RacET mice with T4 resulted

in further increase in Rac expression which was associated with lethal dilated cardiomyopathy

within 1.5 weeks after birth. These cardiomyopathic effects observed in the RacET mice have

been attributed to the alteration in the focal adhesion regulation [73]. In an exciting discovery we

[74] have confirmed the remarkable functional and structural conservation of Rac protein in both

plant and animal kingdoms during evolution and that the constitutively active mutant of Zea

maize Rac D (ZmRacD) is a stronger activator of the oxidative burst than the mammalian one in-

vitro. Our laboratory [70] has further confirmed this conservation in-vivo and showed that over-

expression of a constitutively active cardiac-specific form of ZmRacD gene in the transgenic

mice using the same promoter of RacET mice [73] resulted in cardiac hypertrophy as well as a

moderate decrease in systolic function in older transgenic mice. Besides, the activation of

ZmRacD expression with T4 for 2 weeks led to cardiac dilation and severe systolic dysfunction

in adult transgenic mice. Hypertrophic responses in older ZmRacD mice have been coupled with

increased O2.- production and activation of integrin/mitogen-activated protein kinase (MAPK)

hypertrophic signaling [70]. Additionally, infection of isolated neonatal cardiac myocytes with

an adenovirus expressing a constitutively active form of Rac1 suggested that Rac1 induces

cardiac myocyte hypertrophy mediated through apoptosis signal-regulating kinase (ASK1) and

NF-κB [75] (Figure 3).

Hypertrophy can be a compensatory response to enhance contractility and preserve

cardiac output exclusive of undesirable pathology. Nevertheless, persistent stress can drive this

compensatory process into a decompensated state with reflective alterations in gene expression

profile, contractile dysfunction, and extracellular remodeling [76, 77]. Increased NOX activity

and Rac1 translocation are involved in the transition from compensated hypertrophy to heart

failure [78]. Using the T4-induced ZmRacD transgenic model, we [79] consistently revealed that

Rac-mediated O2.- generation, cardiomyocyte apoptosis, and myocardial fibrosis seem to play a

pivotal role in the transition from cardiac hypertrophy to cardiac dilation and failure. Similarly,

Rac1/NOX-mediated superoxide has been implicated in cardiac dysfunction in endotoxemia

[80], oxidative cardiac injury in a model of renovascular hypertensive cardiomyopathy [81] and

doxorubicin-induced cardiotoxicity [82]. Cardiac injury is also induced by redox-sensitive

activation of mineralocorticoid receptors via Rac1 in cardiomyocytes [83]. Moreover, it has been

reported that in human heart failure, increased NOX-dependent ROS production [8, 84] is

coupled with increased membrane expression and activity of Rac1 [8] (Figure 4).

Rac1/ NADPH Oxidase in Cardiac Arrhythmia

Atrial fibrillation (AF) represents the most common persistent arrhythmia in clinical

practice [85]. Growing evidence illustrates that enhanced atrial oxidative stress might represent a

key factor in promoting and maintaining AF in both animal models and humans. Application of

antioxidants has been shown to prevent and reduce electrical remodeling in pacing-induced AF

in dogs [86, 87] as well as to reduce the risk of early recurrence after successful cardioversion of

persistent AF in human patients [88]. In addition, pacing-induced AF in pigs was found to be

characterized by increased NOX activity and O2.- generation in the left atrium [89]. Right human

atrial appendages of patients with AF have also exhibited increased levels of oxidative markers

such as 3-nitrotyrosine and protein carbonyls [90]. Interestingly, Rac1/NOX activation has been

recently identified as a central contributor in the pathogenesis of AF in old RacET (16 months)

transgenic mice as well as in human patients [91]. The signal transduction pathway involved in

this process is reported to be Angiotensin II stimulated connective tissue growth factor (CTGF)

via Rac1/NOX, causing up-regulation of connexin-43, N-cadherin, and interstitial fibrosis and

thus contributing to atrial structural remodeling [92]. Analysis of younger RacET mice (6

months) showed that atrial over-expression of Rac1 and increased NOX activity were associated

with severe atrial conduction disturbances favoring atrial arrhythmias [93]. Furthermore,

increases in Angiotensin II and phosphorylated signal transducer and activator of transcription

(STAT) 3 were detected in human atrial tissues from AF patients. In cultured atrial myocytes and

fibroblasts, Angiotensin II was found to provoke STAT3 phosphorylation in a Rac1-dependent

way. This was coupled with a direct binding of Rac1 to STAT3, indicating a prospective

molecular mechanism resulting in AF [94] (Figure 5).

Rac1/ NADPH Oxidase in Cardiac Ischemia-Reperfusion Injury

ROS cause oxidative protein modification and act as the major mediator of I/R injury

[95]. There is also substantial evidence that ROS are generated during myocardial I/R [96, 97].

Our group [98] showed that sulfaphenazole protects against myocardial I/R via inhibiting

CYP2C9-driven O2.- and scavenging oxygen free radical. Additionally, we showed that

generation of ROS as a result of acute I/R lung injury may be sufficiently large enough to cause

direct cardiac dysfunction that is independent of injury caused to the myocardium as a result of

regional myocardial IR injury alone [99]. Furthermore, increased NOX expression has been

reported in both animals and humans with myocardial infarction [100, 101]. Moreover,

adenoviral-mediated inactivation of Rac1-GTPase by transient expression of dominant negative

Rac1 (N17Rac1) can protect a wide variety of cells including endothelial cells, vascular smooth

muscle cells, fibroblast and ventricular myocytes from ROS-induced I/R injury [102, 103].

Recently it has been reported that I/R-induced increase in myocardial Rac1 activity in diabetic

mice was associated with increased NOX activity and ROS generation. Deficiency of myocardial

Rac1 or an inhibitor of Rac1 decreased NOX activity and ROS generation in parallel with the

improvement of post-ischemic function and myocardial infarction [34]. In line with these

findings, we recently showed that over-expression of myocardial Rac in adult ZmRacD mice was

associated with increased expression of gp91phox and O2.- production [104]. These molecular

changes render the heart more susceptible to increased post-ischemic contractile dysfunction and

myocardial infarction following acute I/R compared with control mice (Figure 6).

Conclusions and Future perspectives

In summary, growing evidence from both animal and human studies confirm the major

role of NOX and/or Rac1 activation in the development of metabolic syndrome and

cardiovascular diseases. Still, the correlation between Rac1 and NOX in a number of these

diseases as well as the involvement of different NOX isoforms and their interplay needs to be

defined. Additionally, the involvement of Rac1/NOX in some cases has been demonstrated in

cell-based studies in cultured cardiomyocytes, but needs to be reproduced in in-vivo animal

experiments prior to translation to the clinical setting. Clinically, Rac1/NOX has been proposed

as a potentially valuable therapeutic target for metabolic syndrome and cardiovascular diseases.

However, elucidation of the ROS-dependent signal transduction mechanisms, their localization,

and the integration of both ROS-dependent transcriptional and signaling pathways in the

pathophysiology of these diseases seems to be a precondition for effective pharmacological

interventions [105]. Several studies in both animals and humans [8, 78, 79, 91] have shown the

effectiveness of statins in inhibiting Rac1 activation with corresponding improvements in disease

symptoms. Nevertheless, statins have also been shown to affect other signaling pathways in

addition to their cholesterol-lowering effects, which highlights the importance of developing

specific Rac inhibitors, as recently reviewed in detail [106]. For instance, successful in-vivo Rac1

inhibition by NSC23766 has been shown in experimental models of proteinuric kidney disease

and hematopoietic stem cell mobilization [44, 107]. Yet, the effectiveness of this inhibitor in

experimental models of metabolic syndrome and cardiovascular diseases need to be determined.

Since accumulating evidence adds more complexity to Rac1 roles in cardiovascular functions

caution should also be considered during therapeutic strategies using Rac1 inhibitors [108].

Cellular Rac1 specificity is feasibly determined by synchronized actions between the upstream

activators, Rac1, and the downstream effectors. Thus, there is a critical need to entirely outline

Rac1 cell-specific and stimulus-specific signaling axes, instead of Rac1 by itself, in order to

obtain the highest possible selectivity during the therapeutic use of Rac1 inhibitors in the future

[108]. Moreover, large randomized clinical trials are required to validate the beneficial effects of

statins and other novel specific Rac inhibitors in human patients at both acute and chronic

clinical levels.

Statement of author Contributions

ME, HH, MA and MK contributed equally in writing of this manuscript, PJ contributed to the

revised version of the manuscript. All authors reviewed and approved the final version of the

manuscript.

Conflict of interest Statement

We have no conflict of interest.

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Figure legends Figure 1. Involvement of Rac/NADPH oxidase-derived oxidative stress in the pathogenesis

of the metabolic syndrome. Rac1 inhibition with NSC23766 as well as inhibition of NADPH

oxidase with gp47phox deletion (gp47phox-/) or apocyanin could reverse these effects. GEF,

guanine nucleotide exchange factor.

Figure 2. Role of Rac/NADPH oxidase-derived oxidative stress in the development of

vascular remodeling, hypertension and atherosclerosis. Rac1 inhibition with dominant

negative Rac mutant (Rac-DN) or statin, inhibition of NADPH oxidase with gp47phox deletion

(gp47phox-/) or gp91ds-tat, antioxidant such as N-acetyl-L-cysteine, or inactivation of hydrogen

peroxide (H2O2) with catalase could reverse these effects. Ang-II, angiotensin II, GEF, guanine

nucleotide exchange factor, MR, mineralocorticoid receptors, NF-κB, nuclear factor- κB, TF,

tissue factor, ONOO-, peroxynitrite, SOD, superoxide dismutase, VSMCs, vascular smooth

muscle cells, MMPs, matrix metalloproteinases, ECs, endothelial cells.

Figure 3. Role of Rac/NADPH oxidase-derived oxidative stress in the development of

cardiac hypertrophy. Rac1 inhibition with dominant negative Rac mutant (Rac-DN), Rac

deletion (Rac-/-) or statin, inhibition of NADPH oxidase with deletion of gp47phox (gp47phox-/)

or gp47phox (gp91phox-/), or antioxidant such as N-acetyl-L-cysteine, could reverse these

effects. Ang-II, angiotensin II, GEF, guanine nucleotide exchange factor, PAK1, p21-activated

kinase 1, TGF-β, transforming growth factor- β, CTGF, connective tissue growth factor, EGFR,

epidermal growth factor, MAPK, mitogen activated protein kinase, ASK1, apoptosis signal-

regulating kinase, NF-κB, nuclear factor- κB, ERK, extracellular-signal- regulated kinase, JNK,

c-Jun N-terminal kinase.

Figure 4. Role of Rac/NADPH oxidase-derived oxidative stress in the development of heart

failure. Rac1 inhibition with dominant negative Rac mutant (Rac-DN), Rac deletion (Rac-/-),

NSC23766 or statin, inhibition of NADPH oxidase with deletion of gp47phox (gp47phox-/) or

gp47phox (gp91phox-/), or antioxidant such as N-acetyl-L-cysteine, could reverse these effects.

PI3K, Phosphoinositide 3-kinase, GEF, guanine nucleotide exchange factor, MMP, matrix

metalloproteinase, TIMP, tissue inhibitor of matrix metalloproteinase, TNF-α, tumor necrosis

factor- α.

Figure 5. Role of Rac/NADPH oxidase-derived oxidative stress in the development of atrial

fibrillation. Angiotensin-II (Ang-II) receptor blocker, losartan, Rac1 inhibition with dominant

negative Rac mutant (Rac-DN), NSC23766 or statin, could reverse these effects. GEF, guanine

nucleotide exchange factor, STAT3, signal transducer and activator of transcription 3, CTGF,

connective tissue growth factor.

Figure 6. Role of Rac/NADPH oxidase-derived oxidative stress in the development of

myocardial ischemia/reperfusion injury. Rac1 inhibition with dominant negative Rac mutant

(Rac-DN), Rac deletion (Rac-/-), or NSC23766, inhibition of NADPH oxidase with deletion of

gp47phox (gp47phox-/) or gp47phox (gp91phox-/) could reverse these effects. GEF, guanine

nucleotide exchange factor, PAK1, p21-activated kinase 1, E-C, excitation-contraction.