Vascular Pharmacology 63 (2014) 63–70

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Recent developments in the effects of nitric oxide-donating statins on cardiovascular disease through regulation of tetrahydrobiopterin and nitric oxide Sze Ma a,b,c, Christopher Cheng-Hwa Ma d,e,⁎ a

Hong Kong Baptist Hospital, Hong Kong National University Ireland, Ireland c Royal College of Physicians of Ireland, Ireland d NHS Dumfries & Galloway, GMC 7411692, United Kingdom e King's College London School of Medicine, United Kingdom b

a r t i c l e

i n f o

Article history: Received 18 May 2014 Received in revised form 1 August 2014 Accepted 4 August 2014 Available online 17 August 2014 Keywords: Tetrahydrobiopterin Nitric oxide Nitric oxide-donating statins Cardiovascular disease

a b s t r a c t Since the discovery of the importance of nitric oxide (NO) to the human body three decades ago, numerous laboratory and clinical studies have been done to explore its potential therapeutic actions on many organs. In the cardiovascular system, NO works as a volatile signaling molecule regulating the vascular permeability and vascular tone, preventing thrombosis and inflammation, as well as inhibiting the smooth muscle hyperplasia. Thus, NO is important in the prevention and treatment of cardiovascular disease. NO is synthesized by NO synthase (NOS) with tetrahydrobiopterin (BH4) as the crucial cofactor. Many studies have been done to form nitric oxide donors so as to deliver NO directly to the vessel walls. In addition, NO moieties have been incorporated into existing therapeutic agents to enhance the NO bioavailability, including statins. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA), the rate-limiting enzyme of the mevalonate pathway. By inhibiting this pathway, statins lower blood cholesterol and exert their pleiotropic effects through activity in reaction cascades, such as Rho/ROCK and Rac 1/NADPH oxidase pathways. Statins have also been observed to implement their non-lipid effects by promoting BH4 synthesis with increase of NO bioavailability. Furthermore, NO-donating statins in laboratory studies have demonstrated to produce better therapeutic effects than their parent's drugs. They offer better anti-inflammatory, anti-proliferative and antithrombotic actions on cardiovascular system. They also cause better revascularization in peripheral ischemia and produce greater enhancement in limb reperfusion and salvage. In addition, it has been shown that NO-donating statin caused less myotoxicity, the most common side effect related to treatment with statins. The initial studies have demonstrated the superior therapeutic effects of NO-donating statins while producing fewer side effects. Crown Copyright © 2014 Published by Elsevier Inc. All rights reserved.

1. Introduction Since the importance of nitric oxide (NO) to human body has been discovered three decades ago, extensive studies have been done to explore its actions on many organs, including cardiovascular, gastrointestinal, nervous and immune systems [35,47,48]. In the cardiovascular system, NO works as a volatile signaling molecule playing a crucial role in the maintenance of the normal function of the system. It regulates the vascular permeability [22,41] and maintains the vascular tone [40]. It also prevents thrombosis [12], inflammation [13], and inhibits the growth [38] and mitogenesis of smooth muscle cells [63] causing smooth muscle hyperplasia. As a consequence, NO is closely related to ⁎ Corresponding author at: Dumfries & Galloway Royal Infirmary, Dumfries DG1 4AP, United Kingdom. E-mail address: [email protected] (C.C.-H. Ma).

http://dx.doi.org/10.1016/j.vph.2014.08.001 1537-1891/Crown Copyright © 2014 Published by Elsevier Inc. All rights reserved.

cardiovascular disorders, such as hypercholesterolemia, hypertension, and atherosclerosis [9,34,44,68]. Furthermore, cardiovascular disease is the main complication of diabetes that remains the major cause of reduced NO bioavailability. The mechanisms involved lie in alternation of signaling pathway in the endothelial cells [71], enhancement of oxidative stress [74], protein kinase C activation [52,59] and endothelial inflammatory activation [60]. Nitric oxide is synthesized by NO synthase (NOS), with tetrahydrobiopterin (BH4) the vital cofactor of NOS, promoting NOS activity with increased NO production. Various attempts have been made to increase BH4 in the vasculature in order to enhance NO bioavailability, and many trials have also been done to produce nitric oxide donors so as to deliver NO directly to the blood vessel walls [47,48]. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA), the rate-limiting enzyme of the mevalonate pathway. By blocking this pathway, statins lower cholesterol and exert their

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pleiotropic effects through inhibition of important reaction cascades leading to an increase in NO bioavailability (e.g. Rho/ROCK) and inhibition in the formation of reactive oxygen species (e.g. Rac 1/NADPH oxidase) [6, 42,56]. As a result, statins achieve their pleiotropic effects of modulation of immunological reaction, inhibition of thrombotic changes, protection of endothelial function and prevention of vascular muscle cell proliferation. Indeed, statins have also been observed to implement their non-lipid effects by promoting BH4 synthesis with resulting increase of NO bioavailability. Furthermore, NO moieties have been incorporated into some statins with a view to strengthen therapeutic potential while reducing side effects. This review highlights the regulation of NO bioavailability through BH4 and eNOS. It assesses the clinical results of treatment with BH4 and the therapeutic effects of statins through BH4-related mechanisms. Finally, it evaluates the effectiveness of NOdonating statins as compared to their parent drugs.

2. Regulation of nitric oxide bioavailability NO bioavailability is regulated by nitric oxide synthase (NOS). There are three isoforms of NOS: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). eNOS is expressed mainly in the endothelium, promoting the pathways for NO formation. The production of NO depends on two main mechanisms: stabilization of endothelial nitric oxide synthase mRNA (eNOSmRNA) [72,79] and eNOS phosphorylation through Akt/PI3K pathway [23,54] (Fig. 1). These mechanisms are active via mechanical stimuli, including shear stress [23] and biochemical stimuli, such as thrombin [51]. However, bioavailability is limited by its reaction with reactive oxygen species (ROS), including superoxide anion (O− 2 ) and hydrogen peroxide (H2O2). Superoxides can react with NO to form peroxynitrite (ONOO− ). Both peroxynitrite and ROS are potent oxidants that may cause protein nitration and oxidation, lipid peroxidation, enzyme inactivation, and DNA oxidative damage [10,58]. In particular, the oxidation of low density lipoprotein (LDL) produces oxidized LDL, further reducing NO bioavailability by dephosphorylation of eNOS [26,83] and reduction of eNOS mRNA level [65]. The result is endothelial dysfunction and vascular remodeling.

Mechanical smuli eg. Sheer stress OR Biochemical smuli eg. Thrombin

Akt eNOS mRNA stability PI3K

eNOS phosphorylaon

eNOS acvity NO

NO

NO bioavailability 1. Dilataon of blood vessel 2. Inhibion of platelet adhesion to vascular endothelium 3. Inhibion of leucocyte adhesion to vascular endothelium 4. Inhibion of smooth muscle hyperplasia

3. Mechanism of endothelial nitric oxide synthase in nitric oxide synthesis NO is generated by endothelial nitric oxide synthases (eNOS) in the endothelium. eNOS is a homodimer consisting of a reductase domain and oxygenase domain (Fig. 2). Reductase domain maintains the structure of NADPH, FAD and FMN and is responsible for the transfer of electrons to the oxygenase domain. The oxygenase domain contains tetrahydrobiopterin (BH4), molecular oxygen, L-arginine and haem. These two domains are linked by a calmodulin-binding domain [33]. When calcium ions are raised to a certain level, calmodulin affinity to the two domains is enhanced. This causes an increased flow of electrons from NADPH, FAD, FMN and finally to haem in the oxygenase domain of the opposite side. With sufficient substrate L-arginine and cofactor BH4, the electrons will be coupled in the haem with production of NO and L-citrulline as the byproduct [27,37]. BH4 is an important cofactor for the synthesis of NO. It promotes the formation and stability of the dimer, enhancing electron transfer in the dimer and increasing the binding of L-arginine in the haem. In the event of limited BH4 availability relative to dihydrobiopterin (BH2) or eNOS, eNOS becomes unstable and the electron of NADPH uncouples from L-arginine oxidation and superoxide is formed instead of NO, causing eNOS uncoupling and decreased NO bioavailability [46]. Cosentino and Katusic in their earlier study first demonstrated that insufficient BH4 caused dysfunctional eNOS in coronary arteries. This resulted in reduced NO and increased hydrogen peroxide formation leading to oxidative vascular injury [14].

Fig. 1. Schematic diagram showing the pathways leading to NO bioavailability and its effects. Mechanic or biochemical stimuli might activate the Akt/PI3k pathway causing eNOS phosphorylation or eNOS mRNA stability with increased NO bioavailability. Abbreviation: Akt (a serine/threonine protein kinase) , PI3K (phosphoinositide 3-kinase), NO (nitric oxide), eNOS (endothelial nitric oxide synthase).

4. Tetrahydrobiopterin in endothelial nitric oxide synthase coupling BH4 bioavailability is regulated by two ways: (1) de novo synthetic pathway in which the BH4 is synthesized from guanosine triphosphate (GTP). (2) Salvage pathway in which BH4 is formed by the recycle from 7,8-dihydrobiopterin (BH2) (Fig. 3). De novo synthetic pathway is the main process in which sufficient BH4 is produced to maintain NOS coupling [75]. It consists of three steps starting from guanosine triphosphate (GTP) to dihydroneopterin triphosphate, pyruvoyltetrahydrobiopterin and finally to BH4 [1,2,28]. The conversion of GTP to dihydroneopterin triphosphate by GTP cyclohydrolase I (GTPCH) is the rate limiting step, with GTPCH expression regulated by inflammatory stimuli, such as proinflammatory cytokines [39,73]. However, the concentration of BH4 is also regulated through negative-feedback inhibition by BH4 itself in the de novo pathway [29]. In addition, the regulation of BH4 bioavailability is implemented via salvage pathway in which dihydrofolatereductase (DHFR) promotes the recycling of BH4 from BH2. BH2 is formed by oxidation of BH4 [53], and it can cause NOS uncoupling and superoxide formation. DHFR plays a crucial role in eNOS coupling as it maintains the BH4:BH2 ratio. Some

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BH4 offered a new therapeutic option in the management of hypercholesterolemia [15]. Despite the positive outcomes, there are other trials that challenge these findings. Cunnington et al. performed a study on 49 patients with coronary artery disease who were assigned randomly to receive BH4 or placebo for 2 to 6 weeks before coronary artery bypass graft (CABG). The result demonstrated that oral BH4 produced elevated plasma BH4, but it also increased plasma BH2, resulting in unchanged BH4:BH2 ratio. This might be caused by the oxidation of BH4 to form BH2. In internal mammary arteries, BH2 concentration increase was greater than that of BH4. The superoxide was not reduced and BH4 could not produce vasodilatation. The co-administration of BH4 and antioxidant has been attempted to prevent the oxidation of BH4 to BH2, but such a therapeutic effect was short-lived and offered no advantage. The study concluded that oral BH4 therapy had no effect in maintaining normal endothelial function due to systemic and vascular oxidation of BH4 [18]. One explanation is that patients in Cosentino's study were younger and only had one risk factor of hyperchlolesterolemia, whereas the patients in this study had more severe cardiovascular disease backgrounds with multiple risk factors and a wide range of medications. BH4 oral administration might be helpful in early cardiovascular disease but lose efficacy in advanced disease. In view of current results, further studies on patients with different severity of cardiovascular diseases, the modes of BH4 administration and co-administration with other antioxidants are warranted to identify optimal therapeutic effects. 6. Statins in the treatment of cardiovascular disease through tetrahydrobiopterin Fig. 2. Schematic diagram illustrating the structure of endothelial nitric oxide synthase (eNOS) and the flow of electron. The electron flows from NADPH, FAD, and FMN in reductase domain and finally to the haem in the oxygenase domain of the opposite side. With sufficient substrate L-arginine and cofactor BH4, the electron will be coupled in the haem with production of nitric oxide and L-citrulline as the byproduct. Abbreviation: CaM (Calmodulin), BH4 (tetrahydrobiopterin).

recent studies showed that BH4:BH2 ratio is more important in the prevention of NOS uncoupling than the absolute concentration of BH4, since BH2 competes with BH4 for the site in eNOS and is not effective in electron transfer [16,17,77]. Sugiyama et al. found that the insufficiency of BH4 was not sufficient to cause eNOS uncoupling, but BH4:BH2 ratio combined with BH2 concentration determined eNOS coupling [70]. Indeed, Noguchi et al. demonstrated that increased BH2 might cause eNOS dysfunction even in the presence of sufficient BH4 concentration [55]. As a result, the recycling of BH4 from BH2 by DHFR is a vital process in the maintenance of NOS coupling.

Both laboratory and clinical studies have attempted to demonstrate the therapeutic effects of statin through the promotion of BH4 and NO formation. Hattori et al. demonstrated that an increase in guanosine triphosphate cyclohydrolase I mRNA (GTPCH mRNA) led to elevated

Salvage pathway

De novo synthec pathway Guanosine triphosphate (GTP) GTP cyclohydrolase 1 (GTPCH)

Sepiapterin

Dihydroneopterin triphosphate

5. Tetrahydrobiopterin in the treatment of cardiovascular disease Since the beneficial effects of BH4 were demonstrated in the laboratories [2,24,81], attempts have been made to treat patients suffering from cardiovascular disorders with BH4. One study showed that infusion of BH4 could restore the endothelial function in patients with hypercholesterolemia [69]. Other studies revealed that infusion of BH4 could improve vasodilation in patients with Type II diabetes [32] and those with congestive heart failure [66]. Maier et al. also demonstrated that BH4 infusion could prevent vasoconstriction of normal blood vessels in patients with coronary heart disease [84]. While infusion of BH4 appears effective in improving vascular conditions of cardiovascular disorders, the effects of chronic administration of BH4 have yet to be fully tested. Cosentino et al. studied 22 hypercholesterolemic patients (LDL N4.5 mmol) who were randomized to a 4-week course of oral BH4 or placebo. The result identified a combination of raised plasma BH4 levels and restored NO-mediated vasodilatation in patients. Furthermore, BH4 administration maintained the normal NO production and reduced superoxide production in endothelial cells. The study concluded that the reversal of oxidative stress and endothelial dysfunction by oral

Sepiapterin reductase Pyruvoyl tetrahydrobiopterin

Oxidave stress Dihydrobiopterin (BH2)

Tetrahydrobiopterin(BH4)

Dihydrofolate reductase (DHFR) Fig. 3. Diagram showing de novo synthetic pathway and salvage pathway for the regulation of tetrahydrobiopterin (BH4).

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Table 1 Trials comparing the beneficial effects and side effects of nitric oxide-donating statins and their parent drugs. Author Drugs used

Study model

End points

Results

[57]

1. Pravastatin 2. NO-donating pravastatin (NCX 6550) 3. Fluvastatin 4. NO-donating fluvastatin (NCX 6553) 1. Pravastatin 2. NO-donating pravastatin (NCX 6550)

1. PC12 cells 2. Rat aortic smooth muscle cells 3. RAW264.7 murine macrophage cells stimulated with lipopolysaccharides (LPS)

1. cGMP formation in PC12 cells 2. Cell proliferation in rat aortic smooth muscle cells 3. iNOS, cyclooxygenase-2 protein expression, nitrite accumulation in RAW264.7 murine macrophage cells stimulated with LPS

NO-donating compounds: 1. Increased cGMP formation 2. Inhibited cell proliferation in rat aortic smooth muscle cells 3. Reduced iNOS, COX-2 protein expression, and nitrite accumulation in murine macrophage cells Parent drugs had no such effects

1. Normal mice 2. ApoE+/− mice

1.spherocyte adhesion to artery segments 2. ROS production in ApoE+/− spherocytes 3. Relaxation in aortic segments 4. Plasma monocyte chemoattractant protein-1 levels

1. Pravastatin 2. NO-donating pravastatin (NCX 6550) 1. Pravastatin 2. NO-donating pravastatin (NCX 6550)

Human monocytes and macrophages from healthy

1. Cytokine release 2. NF-κB translocation 3. PPARγ protein expression

1. Platelet 2. Tissue factor 3. Mouse model of platelet pulmonary thromboembolism 4. Procoagulation activity by mononuclear cells and macrophages Mouse model of myocardial infarction

1. Platelet activation 2. Tissue factor expression by mononuclear cells 3. Platelet consumption, pulmonary vessel occlusion, and mortality rate 4. Procoagulation activity by mononuclear cells and macrophages

NO-donating pravastatin provided superior effects in: 1. Reduced spherocyte adhesion 2. Decreased ROS production 3. Enhanced relaxation in aortic segments 4. Reduced plasma monocyte chemoattractant protein-1 levels NO-donating pravastatin more effective in 1. Inhibition of cytokine release 2. Inhibition of NF-κB translocation 3. Promotion of PPARγ protein expression. NO-donating pravastatin: 1. Inhibited platelet activation 2. Inhibited tissue factor expression 3. Reduced platelet consumption, pulmonary vessel occlusion, and mortality rate 4. Inhibited generation of procoagulation activity

[20]

[3]

[62]

[21]

[49]

1. Pravastatin 2. NO-donating pravastatin (NCX 6550) 1. Atorvastatin 2. NO-donating atorvastatin (NCX 6560)

1. Hyperlipidaemic mice 2. Rabbit aortic rings 3. RAW264.7 macrophages 4. Platelet-pulmonary thromboembolic mice 5. Platelet adhesion 6. Blood pressure in eNOS knockout mice (LDLR)−/− mice on high fat diet for 16 weeks + femoral artery injury with production of oxygen radicals Polymorphonuclear neutrophils in carotid arteries of normocholesterolemic rabbits 1. Normoglycaemia mice with ischaemic limb 2. Diabetic mice with ischaemic limb 3. eNOS knockout mice

[50]

1. Atorvastatin 2. NO-donating atorvastatin (NCX 6560)

[8]

1. Atorvastatin 2. NO-donating pravastatin (NCX 6560)

[25]

1. Pravastatin 2. NO-donating pravastatin

[43]

1. Atorvastatin 2. NO-donating atorvastatin (NCX 547)

Circulating angiogenic cells (CACs) from volunteers and type-2 diabetic patients cultured in low (LG) and high glucose (HG) conditions

[19]

1. Atorvastatin 2. NO-donating atorvastatin (NCX 6560)

C57BL/6 mice received statins for 2 months (40 mg/kg/day)

1. 2 hour reperfusion 2. 24 hour reperfusion a) 1 hour prior to ischaemia b) 1 hour into reperfusion 1. Serum cholesterol level 2. Vasodilatation in aortic ring 3. iNOS expression and dimer assembly in macrophages 4. Reduction of thromboembolism 5. Platelet adhesion to collagen at high shear 6. Reduction of blood pressure

NO-donating pravastatin provided: 1. Reduced infarct size in 2 hour reperfusion 2. Reduced troponin I, interleukin-1 beta, and mortality rate in 24 hour reperfusion NO-donating atorvastatin: 1. Reduced cholesterol level 2. Increased vasodilatation in aortic ring 3. Reduced iNOS expression and increased dimer assembly 4. Reduced thromboembolism in mice 5. Reduced platelet adhesion to collagen 6. Reduced blood pressure

1. ROS in aorta 2. Interleukin-6 and MMP2 in external wall 3. Lipid-rich lesions in aortic arch 4. Femoral artery (intima/media) thickness

NO-donating atorvastatin caused: 1. Greater reduction in ROS 2. Greater reduction in interleukin-6 and MMP2 3. Greater reduction in lipid-rich lesion in aortic arch 4. Greater reduction in femoral artery intima/media thickness NO-donating atorvastatin produced 1. Reduced neutrophil infiltration 2. Improved inhibition of neutrophil recruitment 3. Reduced monocyte chemotaxis caused by neutrophil supernatants NO-donating pravastatin promoted: 1. Skeletal muscle revascularization 2. Greater limb reperfusion and salvage 3. Increase of EPCs in diabetic mice 4. Recovery of migratory capacity of EPCs in diabetic mice 5. Recovery of angiogenesis in eNOS knockout mice 1. NCX 547 completely restored NO level and functions of HG-cultured CACs; atorvastatin prevented only apoptosis 2. NCX 547 increased Akt activity both in LG-cultured and HG-cultured CACs; atorvastatin enhanced Akt activity only in LG conditions 3. NCX 547 promoted outgrowth and migration of CACs from diabetic patients; atorvastatin had no effect

1. Neutrophil infiltration 2. Neutrophil recruitment 3. Ability of neutrophil supernatants to promote monocyte chemotaxis 1. Skeletal muscle revascularization 2. Limb reperfusion and salvage 3. Circulating endothelial progenitor cells (EPCs) 4. Depressed angiogenesis in eNOS knockout mice

1. Functional assays of CACs from healthy volunteers a) Outgrowth b) Proliferation c) Viability d) Migration e) Senescence f) Apoptosis 2. Akt activity of CACs from healthy volunteers 3. Outgrowth and migration of CACs from diabetic patients 1. Muscle-function assessed by treadmill test 2. Serum creatine kinase activity 3. Citrate synthase activity 4. Muscle histology

Atorvastatin: 1. Reduced muscle endurance 2. Increased creatine kinase activity 3. Reduced citrate synthase activity 4. Caused atrophy of muscle fiber NO-donating atorvastatin did not result in any of the above changes

Abbreviation: ApoE−/− mice (atherosclerotic apolipoprotein E receptor knockout mice), NF-kB (nuclear factor kappa B), PPARγ (peroxisome proliferator–activated receptor γ), (LDLR) −/− mice (low-density lipoprotein receptor−/− mice), MMP2 (matrix metalloproteinase 2).

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BH4 level when human umbilical vascular endothelial cells (HUVEC) were treated with cerivastatin or fluvastatin. Additionally, cerivastatin increased stability of eNOSmRNA [31]. In a recent cell study, Aoki et al. highlighted that fluvastatin enhanced GTPCHmRNA in HUVEC. Statin also increased eNOS phosphorylation at Ser-1177 and Ser-633 through the pathways of PI3-kinase/Akt and protein kinase A (PKA) [7]. In animal models, a study showed that Zuker fatty (ZF) cats had significant deficiency of BH4 leading to endothelial dysfunction. When ZF cats were treated with pitavastatin, the BH4 content increased with eNOS activity raised by 200% and superoxide production reduced by 60%. Normal endothelial function was subsequently restored [67]. In another animal trial, rats with streptozotocin-induced diabetes mellitus were observed. Hyperglycaemia was found to down-regulate GTPCH and BH4 synthesis with subsequent eNOS uncoupling and superoxide production in vascular tissues. Treatment with atorvastatin reversed these changes with improvement of BH4 production and eNOS coupling [78]. A recent study employed a model of rats with 2-kidney-2-clip renal hypertension and demonstrated that GTPCH activity was significantly reduced with a decrease in BH4 production. Treatment with simvastatin markedly improved GTPCH activity and BH4 synthesis [82]. Though both cell and animal studies demonstrated the beneficial effects of statins through the promotion of BH4 synthesis and eNOS coupling, there are limited clinical studies illustrating these effects of statins. Takada et al. recruited 163 patients with cardiovascular disorders and identified a positive correlation between flow-mediated vasodilatation (FMD) and plasma BH4, but a negative correlation between FMD and plasma BH2. As a result, FMD, a marker of endothelial function, had a positive correlation with BH4:BH2 ratio. They further randomized 46 patients into placebo group and treatment group who received a 3-month course of atorvastatin. The result showed that most patients in the treatment group had improved FMD and increased BH4: BH2 ratio, whereas these markers remained unchanged in the control group. They concluded that statin was effective in restoring normal endothelial function and that hypothesized plasma BH4:BH2 ratio might be a useful marker for endothelial dysfunction [85]. However, a number of studies have presented findings that contradict these encouraging results. Antoniades et al. demonstrated that plasma BH4 increased in response to inflammation in normal individuals [4], as well as patients with coronary artery disease [5]. This change in BH4 was accompanied with raised C-reactive protein and reduced vasodilation, indicating that increased plasma BH4 might be a marker of systemic inflammation. In contrast, the increase of vascular BH4 was associated with improved vasodilatation and reduced superoxide, suggesting improvement in eNOS coupling and endothelial function. Indeed, the same team of researchers then demonstrated for the first time the direct effects of statin on human blood vessels. In 492 patients undergoing CABG, those treated with statins had significantly better FMD, improved saphenous venodilatation, and lower superoxide production in internal mammary artery samples. In addition, 42 statinnaïve patients were randomized into placebo group and treatment group of 40 mg/day atorvastatin 3 days before CABG. Results revealed that the statin cohort had significantly improved FMD, along with reduced superoxide production and improved eNOS coupling. Further research demonstrated that the basic mechanisms for improved eNOS coupling are the marked increase of BH4 (3 fold increase) and total biopterins in vascular tissues, caused by the upregulation of GTPCH gene and activity. In contrast, there was reduction in BH4 and total biopterins in the plasma, indicating the action of statins against systemic inflammation. It is interesting to observe that both BH4:BH2 and BH4: total biopterin ratios remained unchanged in plasma and vascular tissue [4]. This finding was contradictory to previous studies that emphasized the importance of these ratios in the diagnosis or normalization of endothelial dysfunction. It seems that the absolute BH4 level and the superoxide production in the vascular tissue might be the more important factors in maintaining normal endothelial functions. To conclude, there

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are strong laboratory and clinical evidences demonstrating the beneficial effects of statins through their action on BH4 synthesis and eNOS coupling. 7. Nitric oxide-donating statins With the effects of nitric oxide on the cardiovascular system demonstrated both in laboratory and clinical trials, various nitric oxide donors have been developed in the past two decades. Diazeniumdiolates (NONOates) and S-nitrosothiols are two of these agents that have been studied extensively and were found to be effective in the prevention of thrombosis and neointimal formation [47,48] leading to clinical application trials including coating for stents [64], polymer films [11] and external surface of sensors [80]. Another approach for providing additional NO consists of incorporating NO moieties into the structure of existing therapeutic agents, including statins (Table 1). Ongini et al. incorporated a NO moiety into pravastatin (NCX 6550) and fluvastatin (NCX 6553), and assessed their anti-inflammatory and antiproliferative effects. The newly formed agents were found to be more potent in inhibiting inflammatory change in RAW264.7 cells and VSMC proliferation in rat aorta than their parent statins [57]. Dever et al. also assessed the anti-inflammatory action of NCX 6550 in atherosclerotic mice. They employed atherosclerotic apolipoprotein E receptor knockout (ApoE −/−) mice that received pravastatin, NCX 6550 or vehicle orally for 5 days. The results revealed that NCX 6550 significantly brought down the spherocyte adhesion to artery segments, reduced ROS production, and improved relaxation of aortic segments. Pravastatin, compared with NCX 6550, only showed inhibition of ROS production without other beneficial effects [20]. Another study evaluated the antiinflammatory effect of NCX 6550 on monocytes and macrophages from healthy human donors and concluded that NCX 6550 offered significantly higher anti-inflammatory activity in these cells than pravastatin [3]. Rossiello et al. studied the antithrombotic effects of NCX 6550 and pravastatin. They found that NCX 6550 significantly inhibited platelet activation and tissue factor expression by mononuclear cells, whereas pravastatin had no such effects. In addition, a mouse model of platelet pulmonary thromboembolism identified that NCX 6550 reduced platelet consumption, pulmonary blood vessel occlusion and mortality rate. NCX 6550 also significantly reduced the generation of procoagulant activity of mononuclear cells and macrophages in mice treated with lipopolysaccharide (LPS). These in vivo changes were not observed upon administration of the parent drug. It was concluded that nitropravastatin offered stronger antithrombotic effects than pravastatin both in vitro and in vivo [62]. Another trial observed the cardio-protective effect of NCX6550 and pravastatin. Using a mouse model of myocardial infarction, NCX-6550 was more effective in reducing the infarct size of lesions and mortality rate than pravastatin, indicating that NCX-6550 provided better cardio-protection. [21]. NO has also been incorporated into atorvastatin forming NOdonating atorvastatin, NCX 6560. In a 5-week course of oral NCX 6560 and atorvastatin in hyperlipidemic mice, Momi et al. revealed that NCX 6560 produced lower cholesterol level, greater anti-inflammatory and antithrombotic effects as compared to atorvastatin [49]. The same team later performed an interesting study involving low-density lipoprotein receptor (LDLR)−/− mice fed with high-fat diet for 16 weeks and sustained injury to the femoral artery resulting in local production of oxygen radicals. Mice were noted to have markedly elevated cholesterol, excessive inflammatory changes, and accelerated atherosclerosis, a condition similar to familial hypercholesterolemia. Treatment outcomes showed that NCX 6560 was significantly superior to atorvastatin in reducing ROS in the aorta, inflammatory markers of interleukin-6 in circulation and matrix metalloproteinase 2 in the external wall. Furthermore, NCX 6560 caused greater reduction in lipid-rich lesions in the aortic arch and femoral artery intima/media thickness. It was concluded that in marked endothelial dysfunction, severe inflammation and accelerated atherosclerosis, even high-dose atorvastatin failed to show

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satisfactory impact on anti-atherogenesis and anti-inflammation, whereas NO-donating atorvastatin offered better results [50]. Indeed, a recent study evaluated the action of NCX 6560 and atorvastatin on polymorphonuclear neutrophils, the first leukocytes infiltrating the inflamed tissue. NCX 6560 had greater inhibition in neutrophil recruitment, and reduced the neutrophil infiltration by 39.5% whereas atorvastatin had no effect on neutrophil content [8]. A study, focusing on the effects of NO-releasing statin on peripheral ischemia, demonstrated that only nitropravastatin (NCX 6550) produced revascularization in the ischemic limbs of normoglycemic or streptozotocin-diabetic mice and caused greater enhancement in limb reperfusion and salvage while pravastatin failed to induce angiogenesis. The study also revealed that high glucose concentration inhibited migration of endothelial progenitor cells in vitro, which was completely recovered with NCX 6550, but only partially reserved with pravastatin [25]. The same researchers further evaluated the effects of NO-donating atorvastatin (NCX 547) on circulating angiogenic cells (CACs) that are important in re-endothelialization and angiogenesis after tissue injury [61]. CACs were obtained from peripheral blood monocytes of healthy individuals and type-2 diabetes mellitus patients, and were cultured in low (LG) or high glucose (HG) conditions. CACs from healthy subjects in HG showed lowered NO, reduced outgrowth, proliferation, and migration of cells while there was increased senescence and apoptosis. NCX 547 restored all these functions whereas atorvastatin only prevented apoptosis. NCX 547 also augmented the outgrowth and migration of CAC from type-2 diabetic patients [43]. To conclude, it appears that NO-donating statins exert superior anti-inflammatory, antithrombotic and anti-proliferative effects in cardiovascular disease compared to their parent drugs. Statins are one of the most common prescribed medications and are considered to be effective, efficient and well tolerated with few side effects [45]. The most common side effect is myotoxicity that includes a spectrum of clinical manifestations of myalgia, myositis and rhabdomyolysis [30,76]. Myalgia is defined as muscle pain with normal creatine kinase (CK) and occurs in 1%–5% of patients. Myositis is defined as muscle pain with abnormal CK and an incidence of 5 per 100,000 person-years. The most serious side effect is rhadomyolysis that presents with muscle ache, CK levels 10 times above the upper limit, and renal insufficiency. Incidence is 1.6 per 100,000 person-years [86]. A recent study attempted to investigate whether NO-donating statin provided a lower incidence of myotoxicity. Atorvastatin or an equivalent dose of NCX 6560 was administered to C57BL/6 mice for 2 months. The results revealed that atorvastatin caused a 6-fold increase in serum CK and a significant reduction in muscle endurance as shown on treadmill test. In addition, histological studies of muscle fibers showed atrophic changes. In contrast, NCX 6560 retained the muscle function, avoided CK increase, and retained normal muscle structure. The study also demonstrated that citrate synthase activity, a biomarker of mitochondrial function, was reduced by atorvastatin and preserved by NCX 6560 [19]. It concluded that NO-donating statin might lower the incidence of myotoxicity. It will be interesting to observe whether further studies confirm these initial findings. As far as laboratory studies revealed, NO-donating statins provided better therapeutic results with reduced complications of myotoxicity (Table 1). 8. Concluding remarks After two decades of research, the pleiotropic effects of statins are well established. Some of the mechanisms related to these effects were found to be crucial in developing new biomarkers and therapeutic agents for cardiovascular disease. The promotion of BH4 synthesis and eNOS coupling in vasculature is considered an important mechanism in managing cardiovascular disease by increasing NO bioavailability. However, the increase in plasma BH4 or plasma BH4:BH2 ratio may simply represent a marker of systemic inflammation. Additionally, chronic administration of BH4 is facing the challenge of oxidation of BH4 to BH2 in circulation before reaching the vessel wall. The

combination of BH4 with different antioxidants may be attempted in the future to achieve better therapeutic effects. Currently, there are both laboratory and clinical evidences confirming the effects of statins in boosting BH4 synthesis and eNOS coupling. The mechanism involves the upregulation of GTPCH activity in the vasculature, leading to increased synthesis of BH4 and NO. Since the pivotal effects of NO have been demonstrated in the prevention and treatment of cardiovascular disease, NO has been incorporated into statins. NO-donating statins are shown to possess stronger anti-inflammatory, antithrombotic and anti-proliferative actions than their parent drugs. In addition, these newly formed drugs offer better therapeutic effects in peripheral ischemia, provide improved cardio-protection, and reduce the risk of adverse effects. 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