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Current Pharmaceutical Design, 2014, 20, 3579-3594 3579

eNOS Uncoupling in Cardiovascular Diseases - the Role of Oxidative Stress and Inflammation

Susanne Karbach1, Philip Wenzel1,2, Ari Waisman3, Thomas Münzel1 and Andreas Daiber1¶

12nd Medical Clinic, Department of Cardiology, 2Center of Thrombosis and Hemostasis and 3The Institute for Molecular Medicine, Medical Center of the Johannes Gutenberg University, Mainz, Germany

Abstract: Many cardiovascular diseases and drug-induced complications are associated with - or even based on - an imbalance between the formation of reactive oxygen and nitrogen species (RONS) and antioxidant enzymes catalyzing the break-down of these harmful oxi-dants. According to the “kindling radical” hypothesis, the formation of RONS may trigger in certain conditions the activation of addi-tional sources of RONS. According to recent reports, vascular dysfunction in general and cardiovascular complications such as hyperten-sion, atherosclerosis and coronary artery diseases may be connected to inflammatory processes. The present review is focusing on the un-coupling of endothelial nitric oxide synthase (eNOS) by different mechanisms involving so-called “redox switches”. The oxidative deple-tion of tetrahydrobiopterin (BH4), oxidative disruption of the dimeric eNOS complex, S-glutathionylation and adverse phosphorylation as well as RONS-triggered increases in levels of asymmetric dimethylarginine (ADMA) will be discussed. But also new concepts of eNOS uncoupling and state of the art detection of this process will be described. Another part of this review article will address pharmaceutical interventions preventing or reversing eNOS uncoupling and thereby normalize vascular function in a given disease setting. We finally turn our attention to the inflammatory mechanisms that are also involved in the development of endothelial dysfunction and cardiovascu-lar disease. Inflammatory cell and cytokine profiles as well as their interactions, which are among the kindling mechanisms for the devel-opment of vascular dysfunction will be discussed on the basis of the current literature.

Keywords: Oxidative stress, nitric oxide synthase uncoupling, redox switches in nitric oxide synthase, inflammatory cells.

INTRODUCTION Oxidative stress was demonstrated to be a hallmark of most cardiovascular and neurodegenerative diseases [1, 2]. The term oxidative stress defines a state with either increased (uncontrolled) formation of reactive oxygen and nitrogen species (RONS) and/or impaired cellular antioxidant defense system (e.g. down-regulation of important antioxidant proteins) with subsequent depletion of low molecular weight antioxidants and a shift in the cellular redox bal-ance. The most common RONS include superoxide radicals, hydro-gen peroxide, hydroxyl radicals, carbon-centered peroxides and peroxyl radicals, nitric oxide radicals (•NO), nitrogen dioxide radi-als, peroxynitrite, hypochlorite and others. Some of these species have a role as cellular messengers and contribute to redox signaling [3-5] such as •NO which acts as an important vasodilator. A direct interaction between •NO and superoxide was proven by the existence of peroxynitrite (ONOO-) that is formed by the diffusion-controlled reaction of •NO with superoxide [6]. Peroxyni-trite is a much more potent oxidant than •NO and superoxide and its contribution to cardiovascular and neurodegenerative disease is meanwhile accepted [2, 7]. Therefore, in many aspects superoxide can be regarded as a direct antagonist of •NO. The superoxide anion (•O2

-) can be formed from different sources such as xanthine oxi-dase, NADPH oxidases, uncoupled NO synthases and the mito-chondrial respiratory chain. The discovery of superoxide dismu-tases (mitochondrial manganese superoxide dismutase (Mn-SOD) and cytosolic/extracellular Cu,Zn-SOD) by Fridovich and cowork-ers in den 1960s [8] suggested that superoxide is formed in the organism and in living cells. Moreover, the existence of SODs im-plied that superoxide is a harmful species involved in pathological processes forcing the organism to express SODs for protection.

¶Address correspondence to this author at the Universitätsmedizin der Jo-hannes Gutenberg-Universität Mainz, II. MedizinischeKlinik und Poliklinik – Labor für Molekulare Kadiologie, Geb. 605 – Raum 3.262, Langen-beckstr. 1, 55131 Mainz, Germany; Tel: +49 (0)6131 176280; Fax +49 (0)6131 176293, E-mail: andreas.daiber@bioredox.com

The first direct evidence for a role of RONS for the regulation of the vascular tone was already provided before the commonly accepted identification of the “endothelium-derived relaxing factor (EDRF)” to be nitric oxide by the observation that EDRF-mediated vasodilation was impaired by superoxide anions and that its func-tion was preserved in the presence of SOD [9]. In these studies Gryglewski et al. used cultured endothelial cells, stimulated them with bradykinine, the supernatants (containing all endothelial me-diators in response to bradykinine) was used to perfuse isolated aortic smooth muscle strips and the vasodilatory activity of the endothelial perfusate was tested by isometric tension recordings. The authors observed that the stability of EDRF, now well known as nitric oxide, is significantly decreased in the presence of super-oxide and preserved by addition of SOD. An indirect clinical proof for endothelial dysfunction triggered by RONS is based on the ob-servation that patients with pronounced improvement of endothelial function (measured by acetylcholine-induced forearm vasodilation) in response to vitamin C infusion were at higher risk for cardiovas-cular events as compared to those patients displaying only minor improvement of endothelial function in response to vitamin C infu-sion [10]. The main conclusion of this study was that patients with increased vascular oxidative stress (making them more responsive to vitamin C) displayed a higher incidence of cardiovascular events. Recent experimental data indicate that inflammatory processes play an important role for the development of endothelial dysfunc-tion and hypertension [11-13] and especially the cytokine interleu-kin 17 plays an essential role in this process [14]. In the present review, the role of inflammatory cells and the phagocytic NADPH oxidase for the induction of vascular oxidative stress, eNOS dys-function (uncoupling) and atherothrombotic events will be dis-cussed in detail (Fig. 1).

THE DAMAGING ROLE OF OXIDATIVE STRESS IN CARDIOVASCULAR DISEASES Harrison and Ohara first described the role of oxidative stress in the progression and pathophysiology of cardiovascular disease in an experimental model of hypercholesterolemia [15, 16]. A lot of pre-

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clinical studies using genetic tools (e.g. knockout mice) followed and clarified the involvement of ROS producing or degrading en-zymes in the onset and progression of cardiovascular disease. They provided the molecular proof of the pathophysiological role of oxi-dative stress in cardiovascular disease: For example, it was shown in mice that the genetic deletion of the NADPH oxidase subunit p47phox almost normalized vascular •NO bioavailability, reduced ROS formation, and improved heart function as well as the survival rate by 20% after myocardial infraction (MI) [17]. Besides, the deletion of the NADPH oxidase subunits p47phox and Nox1 has a protective effect on blood pressure and endothelial function in an-giotensin-II (AT-II)-induced hypertension in mice [18, 19]. Over-expression of Nox1 in these transgenic mice caused a further in-crease in blood pressure [20]. Vice versa, partial deletion of the mitochondrial superoxide dismutase (MnSOD+/-) increased age-dependent mitochondrial oxidative stress and endothelial dysfunc-tion [21]. Also partial deficiency in the mitochondrial superoxide dismutase rendered mice more susceptible to nitroglycerin-induced nitrate tolerance and endothelial dysfunction [22] and deletion of the glutathione peroxidase-1 resulted in an elevated atherosclerotic plaque lesion size in ApoE-/- mice [23]. These data (going in line with several other ones in literature) are a molecular proof of the crucial role of oxidative stress in the development of cardiovascular disease.

THE “KINDLING RADICAL” HYPOTHESIS According to the concept of “kindling radicals” (or also “bon-fire” hypothesis), initial formation of ROS (e.g. from NADPH oxi-dases) triggers further damage such as eNOS uncoupling by differ-ent mechanisms (see “redox switches” below and Fig. 2). The ROS-induced ROS production concept can be extended to almost any kind of source of RONS as almost all of these sources contain “re-dox switches”. Beyond cytoplasmic enzymes, there is clear evi-dence of a cross-talk between mitochondrial ROS formation and NADPH oxidases [24]: mitochondrial ROS can open the mitochon-drial permeability transition pore (mPTP), which is also subject to redox regulation [25]. Upon release of the mtROS to the cytosol,

they can activate the redox-sensitive zinc-finger-like complex in the protein kinase C (PKC) [26] and thereby confer the translocation of cytosolic NADPH oxidase subunits triggering NADPH oxidase dependent superoxide release. Vice versa, NADPH oxidase-derived cytosolic superoxide or peroxynitrite may stimulate mitochondrial ROS formation via opening of the mitochondrial ATP-sensitive potassium channel (mtKATP) [27] leading to changes in the mito-chondrial membrane potential [28, 29]. Examples and the underly-ing mechanisms for this mtROS-NADPH oxidase crosstalk were recently summarized and discussed in detail in three different re-view articles [24, 30, 31]. Other sources of oxidative stress possess similar redox switches (for review see [30, 31]): The conversion of xanthine dehydro-genase (XDH) to the oxidase form (XO) needs for example oxida-tion of critical thiol residues [32, 33] as observed in AT-II induced hypertension [34]. The uncoupling of eNOS (and also other iso-forms) is based on increased formation of oxidants. There exist various reports revealing that eNOS function is improved and un-coupling is reversed when the sources of oxidative stress are either inhibited by pharmacological manipulation (e.g. by PKC inhibitors or NADPH oxidase inhibitors but also AT1-receptor-blockers) [35-40] or genetic deletion of the p47phox or gp91phox subunit leading to a dysfunctional phagocytic NADPH oxidase [41-43]. Similar ob-servations were made when antioxidants were added acutely and at high concentrations to the system (e.g. infusion of vitamin C) [10, 44]. The alliance and interconnection between the different sources of oxidative stress may be the reason why the inhibition of only one source of RONS can be sufficient to completely normalize a car-diovascular disease state. This could for example be demonstrated for the inhibition of xanthine oxidase derived ROS by allopurinol in experimental diabetes, hypertension and pulmonary arterial hyper-tension [34, 45, 46] and for the prevention of mitochondrial ROS formation by mitochondria-targeted antioxidants in hypertension and ischemia/reperfusion [47, 48]. Finally, the exclusive inhibition of the NADPH oxidase by apocynin in diabetes, hypertension and ischemia/reperfusion led to an appreciable normalization of the vascular complications [49-51].

Fig. (1). Inflammatory cells, vascular dysfunction and atherothrombosis. The scheme illustrates the activation of immune cells and recruitment to vascular tissues leading to activation of secondary RONS sources such as NADPH oxidase and uncoupled eNOS, all of which contributes to vascular dysfunction. These processes lead to late-stage cardiovascular complication such as atherosclerosis with plaque formation and thrombosis. The color version of the figure is available in the electronic copy of the article.

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REDOX SWITCHES IN ENDOTHELIAL NITRIC OXIDE SYNTHASE (ENOS) Next to the classical regulation principles of the enzymatic ac-tivity of eNOS (e.g. by calcium/calmodulin, caveolin, HSP90, palmitoylation and myristoylation), there exist other regulatory pathways as phosphorylation and S-glutathionylation, which are directly linked to the formation of redox-active species. These „re-dox switches“ in eNOS confer alterations in enzymatic eNOS activ-ity and may contribute to uncoupling of eNOS (see Fig. 2). In the process of eNOS uncoupling, electrons leak from the transport chain in the reductase domain (from NADPH over FMN and FAD) or directly from the iron-oxy complex during the catalytic cycle. They are then transferred to molecular oxygen to yield superoxide instead of nitric oxide. This development is even more dangerous than the exclusive inhibition of eNOS because the uncoupling trans-forms eNOS from a beneficial to a harmful enzyme [52, 53]. Not only eNOS can be uncoupled and produce superoxide but also the neuronal NOS (type 1) [54-57] and the inducible NOS (type 2) [58-61] can be uncoupled and then produce superoxide themselves. Among the regulatory pathways (“redox switches”) of eNOS, the concept of the oxidative depletion of tetrahydrobiopterin (BH4)is the most fundamental one. BH4 is not only important as steric stabilizer of the NOS dimers. It is also directly involved in the elec-tron transfer from the co-substrate NADPH via the iron-oxy com-plex to the L-arginine substrate. BH4 provides an electron just in time to avoid the decay of the iron-oxy complex under generation of superoxide and iron(III) (see Fig. 3). The resulting BH4

+-radicals are reduced back to BH4 in a subsequent reaction step. As BH4is highly active in redox processes, oxidants easily react with BH4 in 1-electron reactions. Several independent working groups pointed at the substantial evidence for a causative role of BH4 depletion in

Fig. (3). Role of tetrahydrobiopterin (BH4) in prevention of the decom-position of the iron-oxy complex under formation of iron(III) and su-peroxide. Upon activation of molecular oxygen by its binding to the iron(III) of the eNOS catalytic (iron-porphyrin) site and transfer of electrons from the NADPH cofactor via the flavins in the reductase domain, an iron-oxy complex is formed that may decompose under formation of iron(III)and superoxide. BH4 stabilizes this iron-oxy complex by transfer of an elec-tron under formation of a BH4-cation-radical and thereby allows further activation of the oxygen at the iron and subsequent transfer of a hydroxy-group to the guanidino-nitrogen of L-arginine under formation of the inter-mediate NG-hydroxy-L-arginine and reduced BH4. BH4 may participate in a similar fashion in the subsequent reaction step in which L-citrulline and nitric oxide is formed. Adapted from Daiber and Münzel, Steinkopff Verlag Darmstadt 2006 [182]. The color version of the figure is available in the electronic copy of the article.

Fig. (2). Redox switches in endothelial nitric oxide synthase. X-ray structure of human eNOS with the iron-porphyrin (blue), the substrate L-arginine (green), the P450-forming axial iron-thiolate ligand from a cysteine residue (yellow), the cofactor BH4 (purple), the zinc-thiolate complex forming cysteines (red, two from each subunit) and the zinc ion (brown). The red boxes represent the “redox switches” in eNOS triggering regulatory pathways that depend on oxidants and reductants. From Schulz et al., Antioxid. Redox Signal. 2012 [31]. With permission of Mary Ann Liebert Inc. Copyright © 2012. The color version of the figure is available in the electronic copy of the article.

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the process of NOS uncoupling characterized by superoxide forma-tion in the presence of the electron source NADPH [57, 62, 63]: An efficient oxidative degradation of BH4 by peroxynitrite to dihydro-biopterin (BH2) was shown by Milstein and Katusic providing an explanation of how RONS (especially peroxynitrite) may contribute to oxidative uncoupling of eNOS [64]. The understanding of the importance of Vitamin C is important for the recycling or rescue of the •BH4

+ radicals (once BH2 is formed only energy-consuming enzymatic reaction confers reduction to BH4) [65-68], and this led to an attractive explanation for the highly beneficial effects of vita-min C infusion on improvement of the endothelial function in smokers [44] and diabetic patients [69]. First direct evidence of the role of BH4 depletion in eNOS un-coupling and subsequent endothelial dysfunction in vivo was ob-tained in hypertensive mice in 2003 [41]. Soon afterwards, the en-zymatic source for BH4 synthesis in form of the GTP-cyclohy-drolase-1,was identified as an important regulator of eNOS and endothelial function [70]. An overexpression of GTP-cyclohydro-lase-1, improved the endothelial function in atherosclerotic ApoE-/-

mice [71] and vessels from diabetic rats and mice [72, 73]. Besides the GTP-cyclohydrolase-1 dependent de novo synthesis of BH4,there exists the “salvage pathway”, which is of high physiological relevance. It consists of the recycling of oxidized BH2 back to BH4by dihydrofolate reductase [74, 75]. BH4 but not tetrahydroneop-terin (NH4), which indeed shares the same antioxidant properties with BH4without being a cofactor for eNOS led to an improvement of endothelial function in smokers [76, 77]. In line with this, sup-plementation with the BH4 analogue folic acid improved endothe-lial function in humans [78, 79]. Treatment with the BH4 precursor sepiapterin also led to a re-establishment of endothelial function in experimental hypertension and in atherosclerosis [80, 81]. Another direct redox-regulatory pathway for eNOS function is the oxidative disruption of the zinc-sulfur-complex (ZnCys4) in the binding region of the eNOS dimer resulting in a loss of SDS-resistant eNOS dimers. This has first been described by Zou and coworkers for peroxynitrite-mediated oxidation of eNOS [82]. Later they showed that hypochlorous acid also confers disruption of the zinc-sulfur-complex in eNOS [42]. eNOS dysfunction and al-tered eNOS dimer stability by hypochlorous acid was also de-scribed by Keaney Jr. and his coworkers without determination of the zinc content of the enzyme [83]. Wenzel and colleagues also observed this shift in eNOS dimer/monomer ratio in diabetic rats [84]. Although there is no decisive evidence in favor or against a relevant role of the oxidative disruption of the zinc-sulfur-complex for the eNOS uncoupling process, the theory is highly attractive: Peroxynitrite anion (ONOO-) confers high specificity for the oxida-tion of “activated” thiols as thiolate groups that are found in the neighborhood of proton-abstracting amino acids or in zinc-complexes [5]. The reaction of ONOO- with the ZnCys2His com-plex at the active site of alcohol dehydrogenase has been reported to proceed with a kinetic constant of 2.6-5.2*105 M-1s-1[85] and al-ready nanomolar flux rates of superoxide, nitric oxide and per-oxynitrite are able to inactivate the enzyme by oxidative disruption of the ZnCys2His complex associated with zinc release and disul-fide formation [86]. S-glutathionylation is an important redox regulatory mechanism for many enzymes (e.g. mitochondrial aldehyde dehydrogenase [87], sirtuin-1 [88] or SERCA [89]) and was also reported for eNOS. eNOS has been recently described to be adversely regulated and uncoupled (leading to superoxide formation) by S-gluta-thionylation at one or more cysteine residues of the reductase do-main [90]. In a subsequent study, Chen et al. demonstrated a super-oxide-induced thiyl radical formation in eNOS with subsequent intracellular disulfide formation or S-glutathionylation, which both lead to an eNOS uncoupling [91]. Based on the observations by Knorr and colleagues, eNOS S-glutathionylation is largely in-creased in nitroglycerin-treated endothelial cells and aortic tissue

from nitroglycerin-infused rats, probably contributing to eNOS uncoupling and endothelial dysfunction in the setting of nitrate tolerance. This could be prevented by therapy with the AT1-receptor telmisartan [40]. An eNOS S-glutathionylation could also be demonstrated in a rat model of streptozotocin-induced type dia-betes mellitus. Prevention was possible by cotreatment with the organic nitrate pentaerithrityl tetranitrate (PETN) suggesting plei-otropic antioxidant effects based on heme oxygenase-1 (HO-1) induction[92]. Another redox-sensitive regulatory pathway of eNOS is its phosphorylation. Here, we must discriminate 3 different eNOS phosphorylation modifications of relevance: On the one hand´s side the activating phosphorylation at serine1177 is mediated by the Akt pathway. This process is calcium-independent and increases the nitric oxide producing activity of eNOS [93]. Second, the inactivat-ing phosphorylation takes place at tyrosine657, which is arranged by the protein kinase-2 (PYK-2) inhibiting the enzyme without evidence for uncoupling of the enzyme [94]. And third, there exists the inactivating phosphorylation at threonine495 mediated by pro-tein kinase C (PKC), which can also contribute to uncoupling and superoxide production by eNOS [95, 96]. The phosphorylation of eNOS at Thr495 or Tyr657 are activated by oxidative stress. [26, 94, 97]. That means that the phosphorylation at Thr495 and Tyr657 can be regarded as “redox switches” in eNOS. Asymmetric dimethylarginine (ADMA) has often been de-scribed to be the most potent endogenous inhibitor of eNOS [98]. It is still discussed if ADMA itself really evokes uncoupling of eNOS [99]. What is certainly known is that high ADMA serum/plasma levels are a reliable risk marker for cardiovascular events and prog-nosis in patients with cardiovascular disease [100, 101]. Oxidative stress in the vasculature may significantly contribute to ADMA production or to inhibition of ADMA degradation, leading to ADMA concentrations that significantly inhibit eNOS activity [102] or may even uncouple the enzyme and switch it to a superox-ide synthase. It has been described in literature that oxidative stress can increase the expression of PRMTs and thereby lead to increased ADMA-formation [103, 104]. There exists growing evidence that the activity of the ADMA demethylating enzyme (DDAH) is ad-versely regulated by oxidative and nitrosative stress [105]. So the regulation of eNOS activity (and maybe uncoupling) by ADMA seems to be redox-sensitive and may significantly contribute to endothelial dysfunction under oxidative stress conditions (Fig. 4). A recently published work conferred more importance to the concept of ADMA-dependent eNOS regulation and provided a new potential mechanism of endothelial ADMA accumulation despite moderate increases in plasma ADMA levels, which is based on the decreased expression/activity of the y+L amino acid transporters (y+LAT-1) [106]. This concept makes it also easier to understand the so-called L-arginine paradox which consists in the beneficial effect of oral L-arginine on vascular function in patient cohorts despite sufficient L-arginine plasma concentrations for adequate substrate supply to eNOS. According to the recent findings, the export of ADMA in endothelial cells is mediated by y+L amino acid transporters (y+LAT-1 and -2) [107, 108]: These transporters ex-change intracellular cationic amino acids against extracellular neu-tral amino acids and sodium cations and thus provide an active (energy-dependent) efflux pathway for ADMA. Down-regulation of the y+LAT isoforms in cultured endothelial cells by siRNA, lead to an intracellular ADMA accumulation [107, 108]. Consequently, an attractive explanation for the beneficial effects of L-arginine is the export of ADMA via the cationic amino acid transporter (CAT-1) using cationic amino acids (e.g. L-arginine) in exchange in the set-ting of dysfunctional or down-regulated expression of y+LAT [106]. This theory found clinical support by observations in a patient with multiple coronary artery spasm and a genetic defect in the y+LAT expression, causing increased ADMA plasma levels upon admini-stration of high dose L-arginine, most probably by CAT-1/L-

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arginine driven export of ADMA from endothelial cells [106]. These results were actually confirmed by cell culture studies using stable overexpression of CAT-1 [109].

THERAPEUTIC TARGETING OF UNCOUPLED ENOS – “RECOUPLING ENOS” One therapeutic option for eNOS “recoupling” is supplementa-tion with BH4 or its precursors sepiapterin and folic acid, which are cheaper and more stable. BH4 supplementation has proven highly beneficial in many studies on endothelial function such as im-provement in diabetic patients or chronic smokers [110]. Overex-pression or activity enhancement of GTP-cyclohydrolase represents another pathway to increase the BH4 levels as demonstrated for diabetic mice [111]. Under certain conditions the levels of ADMA are increased in endothelial cell, which may contribute to uncou-pling of eNOS, and supplementation with high dose L-arginine may be of great benefit by replacing ADMA or even support its export from endothelial cells by driving the cationic amino acid transport-ers. This may be of great clinical importance since ADMA is a valid predictor of cardiovascular events and increases cardiovascu-lar mortality [100]. Besides these directly eNOS-targeting therapeu-tic interventions any cardiovascular therapy that reduces oxidative stress will contribute to “recoupling” of eNOS as demonstrated for angiotensin-converting-enzyme (ACE) inhibition, AT1-receptor-blocker (ARB) therapy, treatment with statins or antihyperglyce-mics and to a lesser extent treatment with calcium-antagonists or �-blockers. In general, promising strategies to prevent eNOS uncou-pling or to “recouple” an uncoupled eNOS may be based on the increase in cellular antioxidant defense mechanisms. A prominent target for this purpose is the activation of the HO system (most easily of the inducible isoform HO-1). Figure 5 summarizes how induction of HO-1 or the constitutive isoform HO-2 confer protec-tion of the eNOS function in the setting of diabetes mellitus (e.g. by

preventing oxidative depletion of BH4) [112, 113]. Importantly, induction of HO yields low levels of carbon monoxide and biliver-din, further converted to bilirubin, which have anti-atherosclerotic, anti-inflammatory, antithrombotic, vasodilatory and potent antioxi-dant properties. In addition, these products induce Cu/Zn-superoxide dismutase expression, which further contributes to the antioxidant and beneficial profile of HO in diabetic complications. Interestingly, HO products also induce GTP-cyclohydrolase-1, providing a direct link for eNOS regulation [92, 114]. Many cardiovascular drugs have pleiotropic antioxidant proper-ties such as the decrease in NADPH oxidase activity and “recou-pling” of eNOS by ACE inhibitors, ARBs and statins. An interest-ing example of a cardiovascular drug with pleiotropic antioxidant effects is PETN. Once developed in the US, it was abandoned, then used for many years in the former Eastern part of Germany (DDR) and after the reunion of Germany seized the nitrate market. During the last years, it was the best selling nitrate in Germany. PETN is the only organic nitrate in clinical use that is devoid of nitrate toler-ance and endothelial dysfunction [115, 116]. Based on previous observations, PETN is a potent inducer of the intrinsic antioxidant HO-1 system [81] and of extracellular SOD [117]. Even more sur-prisingly, a recent gene array study revealed that PETN, in contrast to GTN, induces a number of cardioprotective genes, whereas GTN induces cardiotoxic genes [118]. Although both compounds are organic nitrates and regarded as nitric oxide donors, they show completely different gene regulation profiles indicating that organic nitrates are not a homogenous class of drugs [116]. Preliminary data indicate that PETN also improves vascular complications in an animal model of type 1 diabetes mellitus. Figure 6 summarizes the beneficial action of several drugs on eNOS coupling state and also introduces different techniques to assess eNOS uncoupling (eNOS-derived superoxide formation).

Fig. (4). Schematic overview of the biochemical pathways related to ADMA. N-methyltransferases utilize S-adenosylmethionine as a methyl group donor and confer the methylation of arginine residues within proteins or polypeptides. Free ADMA is present in the cytoplasm as a product of proteolytic breakdown of proteins and its circulating levels can be detected in human blood plasma. ADMA is a competitive inhibitor of all NOS isoforms replacing the physiological substrate of this enzyme, L-arginine, which contributes to endothelial dysfunction and, as a late consequence, atherosclerosis. The elimination of ADMA from the body proceeds via urinary excretion and, alternatively, via breakdown by the enzyme dimethylarginine dimethylamino hydrolase (DDAH) to citrulline and dimethylamine. Adapted from Böger, Cardiovasc. Res. 2003 [183]. The color version of the figure is available in the electronic copy of the article.

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Fig. (5). The scheme summarizes the simplified mechanisms underlying oxidative stress-induced endothelial dysfunction (and probably nitrate resis-tance) in diabetes mellitus. It should be noted that the oxidative stress concept provides an explanation for a part of diabetic complications and probably repre-sents one important pathological pathway among several. Prevention of diabetic cardiovascular complications by induction of the HO antioxidant system. Key mediators of these beneficial effects are carbon monoxide (CO) bilirubin, extracellular superoxide dismutase (ecSOD), coupling of endothelial nitric oxide synthase (eNOS) by normalization of tetrahydrobiopterin (BH4) levels and decrease in superoxide levels. Adopted from Abraham and Kappas, Pharmacol. Rev. 2008 [112] and Oelze et al., Exp. Diabetes Res. 2010 [113]. Copyright © 2010 Matthias Oelze et al. The color version of the figure is available in the electronic copy of the article.

Fig. (6). Experimental data on eNOS uncoupling and recoupling. (A) Nitric oxide bioavailability was measured indirectly by hemoglobin-nitric oxide com-plex (electron paramagnetic resonance spectroscopy) levels in whole blood samples and by nitrite concentrations (nitric oxide analyzer, n=6) in plasma samples from AT-II infused, hypertensive rats with nebivolol therapy. The electron paramagnetic resonance spectra were averaged from 3 independent measurements. From Oelze et al., Hypertension 2006 [119]. With permission of Wolters Kluwer Health. Copyright © 2006. (B) eNOS uncoupling by oxidative fluorescence microtopography was detected in aortic cryo section from AT-II infused, hypertensive rats with organic nitrate (PETN versus ISMN) therapy. Densitometric quantification of DHE staining was performed in the endothelial cell layer which was extracted from the whole microscope image. A fixed area was used for

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densitometric quantification and the procedure is shown for one representative endothelial cell layer of AT-II treatment group. eNOS uncoupling was previ-ously assessed by the effects of L-NAME on DHE staining [39, 119]. The method of densitometric quantification of endothelial DHE staining was adopted from the protocol of Alp et al. [111]. From Schuhmacher et al., Hypertension 2010 [81]. With permission of Wolters Kluwer Health. Copyright © 2010. (C)Lucigenin-derived chemiluminescence was used to assess vascular ROS formation in intact aortic ring segments from diabetic rats with telmisartan therapy. eNOS-dependent superoxide formation was assessed by substraction of lucigenin signal of L-NAME-treated aortic rings from lucigenin signal without L-NAME and expressed as percentage change of signal based on the L-NAME-free group. Data are shown for control (C), control/telmisartan (C+T), STZ (S), and STZ/telmisartan (S+T) animals. From Wenzel et al., Free Radic. Biol. Med. 2008 [39]. With permission of Elsevier. Copyright © 2008. (D) Levels of eNOS monomer and dimer were determined in aorta from diabetic and atorvastatin treated using Western blot after SDS-PAGE using a 4°C gel and non-reducing conditions. From Wenzel et al., Atherosclerosis 2008 [84]. With permission of Elsevier. Copyright © 2008. (E) S-glutathionylation of eNOS was determined in aorta from nitrate tolerant rats with temisartan therapy by eNOS immunoprecipitation, followed by anti-glutathione staining and normalization on eNOS. Disappearance of the anti-glutathione staining in the presence of 2-mercaptoethanol served as a control. Representative blots are shown at the bottom of each densitometric quantification along with the respective loading control. From Knorr et al., Arterioscler. Thromb. Vasc. Biol. 2011 [40]. With permission of Wolters Kluwer Health. Copyright © 2011. The color version of the figure is available in the electronic copy of the article.

The third generation �-blocker nebivolol had highly beneficial ef-fects on AT-II induced endothelial dysfunction in an experimental model of hypertension [119]. eNOS dysfunction/uncoupling in AT-II treated rats and prevention of this adverse effects by nebivolol therapy can be indirectly measured by the plasma levels of the he-moglobin-nitric oxide complex by EPR, which was decreased in hypertensive rats and increased over control by nebivolol therapy. The reason for this beneficial action of nebivolol is based on the potent inhibition of the NADPH oxidase by the �-blocker, thereby eliminating an important source for the “kindling radicals” (see preceding paragraphs). Similarly, PETN via induction of HO-1 prevents eNOS uncoupling in hypertensive rats as demonstrated by the increase in endothelial superoxide formation by AT-II infusion and normalization of this process by PETN but not isosorbide-5-mononitrate (ISMN) [81]. The method of oxidative fluorescence microtopography using dihydroethidine as a fluorescence probe for endothelial superoxide formation, which is applied here is highly reliable. In combination with L-NAME this assay is highly specific to detect an uncoupled NOS. L-NAME blocks nitric oxide forma-tion from intact eNOS and thereby increases the free, detectable superoxide (DHE signal), by preventing the break-down reaction of superoxide with nitric oxide to yield peroxynitrite. Reciprocally, L-NAME blocks superoxide formation from uncoupled eNOS and thereby decreases the free, detectable superoxide (DHE signal). A very similar technique is based on the modulation of aortic superox-ide release (measured by lucigenin-derived chemiluminescence) by L-NAME in intact vessel segments: The principle of action is just similar to the one explained for oxidative fluorescence microtopog-raphy. We applied this assay to demonstrate increased eNOS un-coupling in aortic tissue from diabetic rats, compatible with the L-NAME-induced decrease in lucigenin ECL signal (blockade of eNOS-derived superoxide formation) [39]. Vice versa, telmisartan therapy normalized the eNOS coupling state, compatible with the L-NAME-induced increase in lucigenin ECL signal (blockade of eNOS-derived nitric oxide formation). Another example for a nor-malization of eNOS coupling state or function by a cardiovascular drug is based on the detection by the eNOS dimer/monomer ratio mentioned before (an indirect read-out), which was decreased in aorta from diabetic rats and normalized by atorvastatin treatment [84]. Finally, the already described eNOS S-glutathionylation rep-resents another indirect read-out for the eNOS coupling or func-tional state and was increased in aorta from nitrate tolerant, nitro-glycerin-treated rats and was normalized by telmisartan therapy [39]. All of these assays to detect eNOS-derived superoxide to assess its coupling state are hampered under conditions of iNOS induction as the iNOS-derived nitric oxide will lead to confusing L-NAME effects. As shown in Figure 7, AT-II infusion in AMPK deficient mice results in severe expression of iNOS and high nitric oxide formation rates (seen by dramatically increased plasma nitrite and aortic nitric oxide EPR signal) [120]. iNOS mimics a functional eNOS with respect to nitric oxide release and especially L-NAME effects (blockade of nitric oxide formation and increase in superox-

Fig. (7). Detection of eNOS-derived superoxide formation in the pres-ence of iNOS. More complicated is the detection of eNOS uncoupling in the presence of iNOS-derived high fluxes of nitric oxide as observed in AMPK deficient mice with AT-II infusion [120]. The infusion of AT-II should result in eNOS uncoupling with subsequent superoxide formation, which is sensitive to L-NAME challenges – blockade of uncoupled eNOS should reduce the superoxide-induced 2-hydroxyethidium (2-HE) signal. In the presence of iNOS, the high fluxes of nitric oxide will mask the eNOS-derived superoxide formation via consumption to yield peroxynitrite. L-NAME will block superoxide formation from uncoupled eNOS but at the same time will inhibit high nitric oxide fluxes from iNOS and thereby the consumption of superoxide to yield peroxynitrite, which will result in the overall increase in superoxide-induced 2-HE signal (also from other ROS sources). Therefore, aortic tissue with uncoupled eNOS and iNOS expres-sion will show similar results as control tissues with respect to the L-NAME sensitive superoxide assay. It is recommended to either use specific inhibi-tors for eNOS and iNOS to characterize the contribution of each isoform or to use indirect methods instead that are based on eNOS specific antibodies (e.g. S-glutathionylation or Thr495/Tyr657 phosphorylation). The color version of the figure is available in the electronic copy of the article.

ide signal). Indicative for the contribution of iNOS to this process were the significantly increased levels of 3-nitrotyrosine-positive proteins. eNOS uncoupling under these conditions is most success-fully documented by indirect methods based on specific eNOS anti-bodies (e.g. dimer/monomer ratio, S-glutathionylation or Thr495/ Tyr657 phosphorylation). Novel strategies to recouple eNOS are based on targeting the drugs to the endothelium. Previous work has demonstrated that administration of extracellular SOD, which directly bind to the

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endothelium, is highly protective in many states of cardiovascular disease [121]. Likewise, deletion of extracellular SOD impaired vascular function and hemodynamics [121]. Therefore, the targeting of extracellular SOD to the endothelium is a promising strategy to prevent endothelial (vascular) dysfunction. In a recent publication, Shuvaev et al. have demonstrated that targeting of SOD but not catalase to the endothelium improved AT-II induced endothelial dysfunction [122]. The antioxidant enzymes superoxide dismutase (SOD) and catalase were conjugated with an antibody to platelet-endothelial cell adhesion molecule-1 (PECAM-1) to ensure endo-thelial binding. The results of this study once more demonstrate that superoxide is the more harmful species in the vascular system than hydrogen peroxide. The covalent binding of antioxidants (e.g. SOD mimics) to heparin may be another promising attempt [123]. This strategy seems to be highly attractive, since the endothelium has heparin-binding sites. The scheme in Figure 8 provides a summary of the oxidant mechanisms that contribute to vascular dysfunction [124]. Possible antioxidant therapeutic interventions are also provided within the

scheme. Despite the fact that most controlled and large clinical trials did not support a beneficial role of oral antioxidant therapy for cardiovascular disease, there is ample evidence that antioxidant therapy may be protective when directed to specific sites and ad-ministrated acutely. In addition, some of the most promising thera-peutic targets have not been exploited so far (e.g. mitochondrial pores or NADPH oxidases) or pleiotropic antioxidant properties of established drugs have not been recognized up to now. Besides the here presented data and the discussed ideas on the development of new synthetic therapeutics, there is the whole field of gene therapy which opens many new pathways with respect to future strategies for antioxidant therapy (e.g. silencing of oxidant producing systems or inducing antioxidant systems) and which is just at its beginning after the discovery of microRNAs and antagomirs.

CARDIOVASCULAR DISEASE, NADPH OXIDASES AND THE IMMUNE SYSTEM As described in the preceding paragraphs, endothelial dysfunc-tion, arterial hypertension as well as other cardiovascular diseases

Fig. (8). Scheme illustrating the mechanisms underlying vascular (endothelial) dysfunction by oxidative stress. Known cardiovascular risk factors (e.g. smoking, hypertension, hyperlipidemia, diabetes) activate the renin-angiotensin-aldosterone-system (RAAS) leading to elevated AT-II levels as well as in-creased endothelial and smooth muscle superoxide (O2

•-) formation from NADPH oxidase activation by protein kinase C (PKC) and from the mitochondria. Superoxide reacts with •NO, thereby decreases •NO bioavailability in favor of peroxynitrite (ONOO-) formation. Peroxynitrite causes uncoupling of endothe-lial NOS due to oxidation of tetrahydrobiopterin (BH4) to BH2 and nitration/inactivation of prostacyclin synthase (PGI2S). Direct proteasome-dependent deg-radation of the BH4 synthase GTP-cyclohydrolase (GTP-CH) further contributes to eNOS uncoupling. Uncoupled NOS produces superoxide instead of •NO and nitrated PGI2S produces no prostacyclin (PGI2) but activated cyclooxygenase-2 (due to increased peroxide tone) generates vasoconstrictive prostaglandin H2. Inhibition of smooth muscle soluble guanylylcyclase (sGC) by superoxide and peroxynitrite contributes to vascular dysfunction as well as increased inacti-vation of cyclic GMP (cGMP) by phosphodiesterases (PDE) and oxidative stress increases the sensitivity to vasoconstrictors such as endothelin-1 (ET-1). Mitochondrial ROS formation is modulated by oxidative activation of ATP-dependent potassium channels (KATP) leading to altered mitochondrial membrane potential and permeability. Upon uncoupling of mitochondrial respiratory complexes, the mitochondrial permeability transition pore (mPTP) may be oxida-tively opened allowing mtROS to escape to the cytosol activating the PKC-NADPH-Ox system. Modified from Münzel et al., Circ. Res. 2005 [115]. With permission of Wolters Kluwer Health. Copyright © 2011. Reproduced from Chen, Chen, Daiber, Faraci, Li, Rembold and Laher, Clinical Science 2012 [124]. With permission of Portland Press. Copyright © 2011. The color version of the figure is available in the electronic copy of the article.

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are usually accompanied and at least partially mediated by in-creased levels of oxidative stress in form of increased RONS for-mation: An elevated RONS formation by NADPH oxidases or by the uncoupled eNOS goes in hand in hand with an insufficient neu-tralization of RONS leading to a dysequilibrium between RONS formation and degradation. A recent advance in the understanding of the development of endothelial dysfunction consists of the integration of the immune system (e.g. inflammatory cells) in this concept (Fig. 9): Besides endothelial cells and vascular smooth muscle cells, inflammatory cells such as neutrophil granulocytes and macrophages (myelo-monocytic cells) but also T-cells and dendritic cells provide func-tional NADPH oxidases and are capable to produce RONS or at least to activate phagocytic cells leading to an “oxidative burst” of the blood [11, 125]. Different immune cells have been reported to contribute to the development of cardiovascular disease but since they interact with each other, the individual impact of each cell type on the development of cardiovascular disease remains elusive. It has been shown that mice without B- and T-cells (RAG-1-/-)mice show a less pronounced superoxide production and a less severe development of hypertension in response to AT-II [11]. Ab-lation of myelomonocytic cells also attenuated the AT-II-induced blood pressure increase and ROS/RNS production [13]. As a possi-ble link to these important observations, it has been described that immune suppressive treatment of patients with rheumatoid arthritis or psoriasis leads to reduction of systolic blood pressure [126], conferring more clinical impact to this new hypothesis of immune system triggered vascular dysfunction.

MYELOMONOCITIC CELLS IN HYPERTENSION Macrophages/monocytes and especially neutrophil granulocytes are capable to produce large amounts of ROS/RNS within a short period of time for host defense within the innate immediate immune response [127] – this process is called “oxidative burst”. Both, neu-

trophils and macrophages/monocytes can be activated and stimu-lated by AT-II over angiotensin receptor type 1 (Agtr1). AT-II leads to the recruitment of CD45+ leucocytes to the aortic wall [14] – and one part of these cells consists of infiltrating myelomonocytic cells [13]. By selective ablation of the LysM-positive myelomonocytic cells with low-dose diphteria toxin using the CreLox approach, the AT-II induced endothelial dysfunction and hypertension as well as the AT-II induced oxidative burst in the blood was suppressed. Vascular dysfunction was reestablished by adoptive cell transfer of monocytes but not of neutrophils [13]. The changes in ROS forma-tion were accompanied by an up-regulation of the catalytic subunit of the phagocytic NADPH oxidase Nox2 under ATII and by a decrease of NOX2 in the ATII treated mice depleted of the myelomonocytic cells [13]. Neutrophils are also implicated to be important in the develop-ment of endothelial dysfunction: Neutrophil granulocytes in spon-taneously hypertensive rats (SHR) provided an elevated oxidative burst (whereas the phagocytic potential was comparable), an in-creased iNOS expression and an enhanced myeloperoxidase (MPO)-catalysis [128]. In addition, a subendothelial MPO accumu-lation by infiltrated neutrophils was described as a major sink for oxidative nitric oxide depletion with subsequent development of endothelial dysfunction [129-131]. This prompts the speculation that neutrophils are at least partially involved in the development of oxidative stress, vascular dysfunction and hypertension. Aside from that, elevated concentrations of MPO in peripheral blood, which is the most abundant enzyme stored in the granules of neutrophils [132] and directly related to neutrophil number [133], are connected to coronary artery disease [134, 135]. It was shown that MPO se-rum levels in patients with ACS predict an increased risk for subse-quent cardiovascular events and by this way extend the prognostic information we get with the help of traditional biochemical markers [129]. There is in addition striking evidence that the NOS inhibitor asymmetrical dimethylarginine (ADMA), which is associated with

Fig. (9). Scheme illustrating the role of the immune system in the development of endothelial dysfunction. Dendritic cells in the vascular wall (possibly presenting neo antigens as answer to hypertensive stimuli like in atherosclerosis [163]) produce IL-6, IL-1 and TGF-�. Thus CD4 T cells are activated and switch to Th17 cell. IL-17A then activates T cells themselves but also endothelial cells, smooth muscle cells and macrophages. Endothelial cells can produce IL-6 and also G-CSF when activated. Macrophages secrete IL-6 and TNF-� as answer to IL-17A contributing to neutrophil activation and recruitment to the site of inflammation. Processes like this can take place in all kinds of locations and also in the vascular wall leading to an upregulation of inflammatory cells in the vasculature. Retraced from Iwakura et al., Immunity 2011 [138]. The color version of the figure is available in the electronic copy of the article.

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endothelial dysfunction, leads to an elevated adhesion of neutro-phils to endothelial cells and to an increased superoxide generation and release of MPO by neutrophils [136].

INTERLEUKIN-17A AND IL-6 IN ENDOTHELIAL DYS-FUNCTION IL-17A is an important cytokine which is responsible for neu-trophil recruitment and macrophage activation [137, 138]. IL-17A is mainly produced by the T helper 17 subset of CD4+ T cells (Th17) and by �� TCR expressing T cells (�� cells). IL-6, which is produced by the innate immunity cells (e.g. dendritic cells, mono-cytes, macrophages, mast cells, B-cells as well as endothelial cells, keratinocytes and fibroblasts), is an essential differentiation factor of Th17 cells [137]. Furthermore, IL-6 is secreted together with TNF-�, IL-1 and IL-12 by macrophages which are triggered by IL-17A, being part of an inflammatory amplification loop [138]. Thus, IL-6 is located upstream, but also downstream of IL-17A. IL-17A has been described to be involved in vascular inflammation of athe-rosclerosis and hypertensive disease [139]. Madhur et al. demon-strated that IL-17A plays a leading role in the development of AT-II-induced vascular dysfunction: The aortas of IL17A deficient mice showed only a mild impairment of the endothelial function under AT-II in comparison to AT-II infused control mice. In addi-tion, superoxide production was reduced in IL-17A-/- [14]. Madhur et al showed that AT-II increases the IL-17A production of T-cells and that chronic AT-II infusion over 4 weeks led to a reduced T-cell infiltration into the aortic wall of IL-17A-/- mice in comparison to control mice with the same treatment. Although the initial blood pressure increase after 4 weeks of AT-II treatment was similar in IL-17A-/- and control mice, IL-17A-/- deficiency ameliorated the elevated blood pressure levels averaging 30 mm Hg lower as com-pared to AT-II infused control mice. It was shown before using IL-6 knock-out mice that IL-6 is important for the onset of AT-II-induced endothelial dysfunction and superoxide production [140]: AT-II-triggered endothelial dysfunction in carotid arteries from IL-6-/- mice was less pronounced as compared to AT-II-treated control mice and a similar observation was made for aortic superoxide levels measured by lucigenin enhanced chemiluminescence [140]. Increased levels of IL-17 were found in patients with myocar-dial infarction respectively and patients with unstable angina [106, 139, 141], as well as in patients with hypertension [14] as compared to healthy controls and patients with stable angina. Overall, plasma IL-6 levels were described to correlate positively with the progres-sion of cardiovascular disease [142]. IL-17A is not only involved in neutrophil and macrophage acti-vation and recruitment but also triggers the pro-inflammatory re-sponse and ROS-production of the vascular smooth muscle cells (VSMC) [143]: IL-17A leads to an increased ROS-production in murine aortic VSMC which could be abolished by the NADPH-oxidase inhibitor apocynin. Treatment with siRNA against Nox2 led to a significant reduction of the IL-17A stimulated ROS-formation demonstrating that Nox2 plays an important role in the IL-17A induced oxidative stress in VSMCs. In addition, IL-17A treatment of VSMCs led to an increase of IL-6, Granulocyte Col-ony-Stimulating Factor (G-CSF) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) in the supernatants of the cell culture, and all of these cytokines play an important role in neutrophil and macrophage activation and recruitment [143]. Even more importantly, IL-17 was shown to be able to induce cell death in human endothelial cells suggesting its involvement in human acute coronary syndrome (ACS) by contributing to plaque destabilization [144]. It has been demonstrated that IL-17A induces disruption of the blood-brain-barrier by induction of NADPH-derived production of ROS by the human brain endothelial cell layer. This led to a down-regulation of the tight junction molecule occludin between the endothelial cells and by this mechanism to a disruption of the blood-brain-barrier [145]. This could be avoided

either by application of an IL-17A blocking antibody or by inhibi-tion of ROS formation [145]. Finally, IL-17 is an important activa-tor of the endothelium, which leads to an induction of endothelial adhesion molecules favoring the adhesion and transmigration of neutrophils [146].

T CELLS IN HYPERTENSION Patients treated for cancer with infusion of autologous, acti-vated lymphocytes from the peripheral blood exhibited an increased blood pressure within the first hours of infusion pointing to a possi-ble role of T cells in the development of hypertension [147]. As summarized above IL-17A, produced by Th17 and �� T cells, plays an important role in the development of endothelial dysfunction. In accordance with these observations, Guzik et aldemonstrated that the elevation of blood pressure caused by AT-II was markedly reduced in mice without B and T cells (RAG-1-/-

mice) [11]. The development of vascular dysfunction, vascular hypertrophy as well as vascular superoxide production was likewise suppressed in AT-II-treated RAG-1-/- mice compared to AT-II-treated control mice. Adoptive cell transfer of functional T-cells (but not of B cells) restored the hypertensive response to AT-II as well as the AT-II-induced increase in vascular superoxide produc-tion. Transfer of T cells lacking the AT-II receptor type 1 (Agtr1) or a functional NADPH oxidase only partially reduced the AT-II-mediated hypertensive response [11]. There is an infiltration of T cells to the aortic adventitia and the periadventitial fat under AT-II-treatment which could be proved by FACS-analysis of the aortic vessel [11]. RAG-1-/- mice had a similar reduction in the hyperten-sive response and the vascular superoxide production compared to control mice when blood pressure was induced with the DOCA-salt model, which shows that the reduced hypertensive response in RAG-1-/- mice was not specific to AT-II. Blood-derived T cells themselves produce AT-II when treated with anti-CD3 [148]. This could be prevented by application of the angiotensin-converting enzyme inhibitor (ACE-inhibitor) perindo-pril. Furthermore T cells seem to express the angiotensin-conver-ting enzyme, renin and both the AT-II receptor type 1 and type 2. Thus, they provide all enzymes of the renin-angiotensin-aldosterone system (RAAS) – meaning that T cells have an own, endogenous RAAS [148, 149]. T cells produce TNF-� as response to exogenous and endogenous AT-II. Endogenously-produced AT-II induces ROS-production in T cells most likely via activation of NADPH-oxidases, and ROS themselves stimulate T cells to produce TNF-�[148]. Treatment with etanercept, the soluble TNF-� receptor anti-body, during AT-II-infusion led to a reduction of AT-II-mediated vascular superoxide production and prevented hypertension [148, 149]. The latter may provide a link between thrombosis and hyper-tension and thereby the basis for atherothrombotic events in CAD patients. The role of T cells in the development of pulmonary arterial hypertension remains ambiguous: RAG-1-/- mice did not develop pulmonary artery hypertension induced by monocrotaline in con-trast to wild type mice where monocrotaline leads to pulmonary vascular endothelial cell damage followed by a perivascular in-flammatory response, also with infiltration of CD4 T cells. Transfer of CD4+ T cells into RAG-1-/- mice via adoptive T cell transfer restored the effect of monocrotaline causing vascular damage in the pulmonary artery [150]. However, it has been reported that athymic nudes rats which lack T cells more easily develop severe pulmonary hypertension when treated with the vascular endothelial growth factor receptor blocker than euthymic rats [151]. In this case, injec-tions of splenic immune cells provided protection [151]. So we are here probably faced with a double-edged problem. Regulatory T cells (Tregs) are CD4+ T cells that express con-stantly the IL-2 receptor CD25 and the transcription factor X-linked forkhead/winged helix (Foxp3). They are generally described to suppress innate as well as adaptive immune responses [152, 153]

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and they also seem to confer improvement in hypertension: The AT-II-induced hypertension was significantly normalized in mice with adoptive transfer of CD4+ CD25+ T cells (and not by the adop-tive transfer of CD4+ CD25- T cells) [154]. Also the AT-II-evoked oxidative stress of the aortic vessel was reduced by adoptive Treg transfers shown by DHE staining and normalization of the NADPH oxidase activity [154]. This fits to the discovery that IL-10 secreted by Tregs attenuates NADPH oxidase activity [155]. The number of Tregs detected in the aorta was very low in control mice and not altered by AT-II-treatment nor by adoptive transfer of Tregs or effector T cells. But there were Foxp3+ cells detectable in the renal cortex of control mice shown by immunofluorescence – and the number was decreased under AT-II treatment and significantly elevated in AT-II-treated mice with adoptive Treg transfer [154]. In line with this the adoptive transfer of Tregs also prevented the al-dosterone-induced vascular injury [156].

DENDRITIC CELLS (DCS) IN HYPERTENSION As the T cell response is controlled by antigen-presenting cells (APCs) like dendritic cells (DCs), it may be assumed that DCs are also involved in the development of endothelial dysfunction. DCs are responsible to prime naïve T cells [157, 158] and to initiate the adaptive immune response after being activated themselves by in-nate stimuli. Among various mechanisms, naïve T cells are acti-vated by the binding of the T cell CD28 to the B7 ligands CD80 and CD86 on DCs [159]. It was shown that the percentage of CD11c+ cells expressing the activation marker CD86 was upregu-lated in the spleen and lymphnodes of mice treated with AT-II which could point to a possible maturation of DCs under AT-II [160]. It had been shown previously that DCs have an own RAAS and that AT-II can modulate functionality and activation of DCs [161]. Blockade of CD28 interaction with the B7 ligands by cyto-toxic T lymphocyte antigen 4 immunoglobulin (CTLA4-Ig) led to a significant reduction of the AT-II-induced hypertension [160]: Be-sides, there was no increased superoxide production in aortas from mice treated with CTLA4-Ig under AT-II whereas in aortas from AT-II-treated mice that had not been treated with CTLA4-Ig the superoxide production was 3-fold increased. Also the aortic infiltra-tion with T cells under AT-II was reduced by CTLA4-Ig treatment showing the importance of the interaction between T cells and DCs in the development of AT-II-induced endothelial dysfunction [160]. In parallel it was shown that aldosterone increased the capacity of DCs to activate CD8+ T cells and aldosterone-treated DCs pro-moted a polarization of CD4+ T cells to Th17 cells [162] which are important in the development of hypertension as previously de-scribed. It is being discussed that hypertensive stimuli could lead to the formation of neoantigens like in atherosclerosis [163] which APCs like DCs present to T cells for induction of T cell activation [160]. Activated T cells then are involved in the development of hypertension as already described above.

B CELLS IN HYPERTENSION Up to now, only very little is known on the involvement of B cells in the development of arterial hypertension and endothelial dysfunction. Aside from the classical arterial hypertension, B cells have been described to contribute to the development of hyperten-sion in response to placental ischemia during pregnancy [164] and that peripheral activated B cells are involved the pathogenesis of idiopathic pulmonary arterial hypertension [165]. As only the adoptive cell transfer of T-cells but not of B cells could restore the hypertensive response and superoxide production to AT-II in mice RAG-1-/- mice which lack per definition B and T cells [11], B cells do not seem to be as important in the develop-ment of hypertension as T cells. But it has nevertheless been shown that B cell deficiency reduced and even abolished the cold-induced blood pressure elevation [166] and that there is an up-regulated immunoglobulin formation and secretion in B cells from hyperten-

sive patients [167]. Therefore, it could be interesting to further elu-cidate the role of B cells in the development of hypertension.

CHRONIC AUTOIMMUNE DISEASES ASSOCIATED WITH CARDIOVASCULAR DISEASE Several chronic autoimmune diseases have been reported to be linked with an increased risk for cardiovascular events: Rheumatoid arthritis, systemic lupus erythematodes and also severe psoriasis have been described to be connected to cardiovascular disease [168-171]. Psoriasis has even been described to be a new risk factor in-dependent of the classical cardiovascular risk factors [172]. Con-cerning the elevated cardiovascular risk of patients with arthritis the European League against Rheumatism has published recommenda-tions for cardiovascular risk management in inflammatory arthritis (including rheumatoid arthritis, psoriatic arthritis and ankylosing arthritis) [173]. Preceding the cardiovascular events, a reduced vascular func-tion was associated with these diseases: So, the carotid intima-media thickness in patients with early rheumatoid arthritis did not differ from the control patients at the beginning of the disease – but 18 months after the first evaluation there was already a significant increase in the intima-media thickness compared to control group [174]. It was also shown that psoriasis patients have an impaired endothelial function measured by flow-mediated dilatation and an increased intima-media thickness of the carotid artery compared to healthy controls [175]. The existing connection between the autoimmune diseases mentioned above and cardiovascular disease is certainly depending on many different factors (also differing in the existing primary diseases. All these diseases present the situation of a chronic in-flammation with at least locally elevated cytokine levels and acti-vated inflammatory cells: In the pathogenesis of psoriasis, the IL-17/IL-23 axis plays a pivotal role [176-178]. Also in patients with systemic lupus erythematodes, IL-17A production is, besides other cytokines, increased [179]. In rheumatoid arthritis IL-6, TNF-� and also IL-17A are of big importance [180, 181]. All these cytokines are, as mentioned above, of relevance in the development of endo-thelial dysfunction. Thus, this could be the link between the devel-opment of cardiovascular disease and the described chronic auto-immune diseases [168, 170]. However, further clinical studies and mechanistic analyses are required.

CONCLUSION AND CLINICAL IMPLICATIONS Cardiovascular disease, the most important reason for death in industrial nations, is a multifactorial complication involving a lot of different mechanisms as uncoupling of eNOS, reactive oxygen species formation, adverse calcium homeostasis and signaling asso-ciated with a deleterious phosphorylation pattern as well as futile counter-regulatory mechanisms at the humoral and cellular/tissue structure level (to mention some of the important contributors). According to more recent data, also immune cells and inflammatory mechanisms play an essential role in cardiovascular disease. The present review wants to highlight the link between the immune system and cardiovascular disease which may translate to more integrative therapies using multidisciplinary expertise and simulta-neously curing the underlying multifactorial complications.

CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENTS We thank Margot Neuser and Thilo Weckmüller for graphical assistance. The present work was supported by generous financial support by the Federal Ministry of Education and Research (BMBF 01EO1003 to P.W., T.M. and A.D.). Susanne Karbach holds a grant

3590 Current Pharmaceutical Design, 2014, Vol. 20, No. 22 Karbach et al.

of the “Margarethe-Waitz-Stiftung” and a MAIFOR-grant. All authors are supported by the “Stiftung Mainzer Herz”.

ABBREVIATIONS ADMA = Asymmetric dimethylarginine ACE = Angiotensin-converting-enzyme Agtr1 = Angiotensin receptor type 1 AT-II = Angiotensin-II BH4 = Tetrahydrobiopterin CAD = Coronary artery disease CTLA4-Ig = Cytotoxic T lymphocyte antigen 4 immunoglo-

bulin DDAH = Dimethylarginine dimethylamino hydrolase DC = Dendritic cell DHE = Dihydroethidine DOCA = Deoxycorticosterone acetate ECL = Enhanced chemiluminescence EDRF = Endothelium-derived relaxing factor EPR = Electron spin resonance eNOS = Endothelial nitric oxide synthase FAD = Flavin adenine dinucleotide FMN = Flavin mononucleotide Foxp3 = Transcription factor X-linked forkhead/winged

helix �� cells = Gamma delta T cells G-CSF = Granulocyte colony-stimulating factor GM-CSF = Granulocyte-macrophage colony-stimulating

factor HO = Heme oxygenase (HO-1, inducible isoform;

HO-2, constitutive isoform) IL-6 = Interleukin-6 IL-17A = Interleukin-17A iNOS = Inducible nitric oxide synthase L-NAME = NG-nitro-L-arginine methyl ester MI = Myocardial infarction mPTP = Mitochondrial permeability transition pore nNOS = Neuronal nitric oxide synthase Nox = NADPH oxidase PETN = Pentaerithrityl tetranitrate PKC = Protein kinase CPYK = Protein tyrosine kinaseRAG-1-/- mice = Recombination activating gene-1 knockout

mice RONS = Reactive oxygen and nitrogen species RNS = Reactive nitrogen species ROS = Reactive oxygen species SOD = Superoxide dismutase SHR = Spontaneously hypertensive rats Th17 = T helper 17 subset of CD4+ T cells TNF-� = Tumor necrosis factor �VSMC = Vascular smooth muscle cells XDH = Xanthine dehydrogenaseXO = Xanthine oxidase

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Received: July 19, 2013 Accepted: October 21, 2013