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Nitric Oxide Signalling in Vascular Control and Cardiovascular Risk

Written By

Annette Schmidt

Submitted: May 5th, 2011 Published: March 14th, 2012

DOI: 10.5772/34801

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1. Introduction

Nitric oxide – a free radical molecule – has been known for many decades, but only since its recognition as endothelium-derived relaxing factor (EDRF) the interest in the molecule has exponentially increased (Moncada, 1991). At the present time NO is an important messenger that regulates numerous functions and also participates in the pathogenesis of various diseases (Lloyd-Jones & Block, 1996). NO is generated from the conversion of arginine to citrulline in a multistep oxidation process by the NO-synthase (NOS), a NADPH-dependent enzyme that requires Calcium-Calmodulin, Flavinadeninedinucleotide, Flavinmononcleotide and Tetrahydro-L-biopterin as cofactors (Förstermann et al., 1994). Three isoforms of NOS have been identified. All isoenzymes, the neuronal NOS (nNOS), the inducible NOS (iNOS) and the endothelial NOS (eNOS) (Liu & Huang, 2008), are homodimers with subunits of 130 – 160 kDa. As major signalling molecule of the vascular system NO is generated by the constitutively expressed eNOS.


2. Endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) function

2.1. eNOS

The endothelium maintains the balance between vasodilation and vasoconstriction. NO generated by eNOS acts via cGMP-dependent pathway in a paracrine manner on neighbouring smooth muscle cells (SMC) diffusing radially from the production site. NO has a half-life of only a few milliseconds in vivo (Tab. 1) and rapidly reacts with iron of the heme moiety in the active site of the enzyme guanylate cyclase, stimulating it to produce the intracellular cGMP that in turn enhances the release of neurotransmitters resulting in SMC relaxation and vasodilation (Fig. 1). Acting via cGMP-independent pathways it is used in part to S-nitrosylation of intracellular or extracellular proteins (Castel & Vaudry, 2001; Mallis et al., 2001; Sun et al., 2001) or by inhibiting intraendothelial generated superoxide anions (Clancy et al., 1992).

2.2. NO functions

Beside its role as vasodilator various other activities of NO have been described: (I) NO prevents the expression of cell adhesion molecules thereby preventing leukocytes/monocytes adhering to vascular endothelium and their immigration into the arterial wall.

Compound Blood/Plasma Levels, nmol/L T1/2
Nitrate 20 000 - 50 000 5 - 8 hours
Nitrite 100 - 500 1 - 5 minutes
NO <1 1 - 2 milliseconds
Hb-NO <1 - 200 15 minutes
S-nitroso-Hb <1 - 200 --
S-nitroso-albumin 1 - 200 --

Table 1.

Basal blood/plasma levels and half-lifes of some NO-related compounds. Values are approximated from studies in human. For Hb-NO, S-nitroso-Hb and S-nitroso-albumin, no firm agreement about normal values has been reached, and reported values vary greatly. T1/2 for Hb-NO is from pig experiments, values for S-nitroso-Hb and S-nitroso-albumin are unknown (from J.O. Lundberg and E. Weitzberg, Arterioscler Thromb Vasc Biol, 2005;25:915-922).

Figure 1.

Nitric oxide signalling axis as therapeutic target in cardiac and vascular disorders. The endothelial eNOS catalyses the formation of NO from L-arginine through two sequential monooxygenation steps. The nitrogen atom of NO is derived from the guanodinogroup of the L-arginine side chain and the oxygen atom of NO derived from molecular oxygen. The cGMP generation in the vascular smooth muscle cell is catalysed by the soluble guanylate cyclase stimulated by the nitric oxide generated by the adjacent endothelial cell.

The monocytes accumulated in the arterial wall can promote local expression or activation of matrixmetalloproteases, which decrease the strength of the cap by degrading collagen and other extracellular matrix components. Furthermore, activated macrophages kill neighbouring SMC by lytic damage leading to necrosis or by inducing apoptosis (Kockx et al., 1996, 1998). (II) NO reduces the influx of lipoproteins into the vascular wall and inhibits LDL oxidation. (III) NO inhibits DNA synthesis (Förstermann et al., 1994) and proliferation of SMC (Li & Förstermann, 2000; Li et al., 2002a). (IV) NO released towards the vascular lumen is a potent inhibitor of platelet aggregation and adhesion (Busse et al., 1987; Radomski et al., 1987). (V) NO can react with superoxide anion O2 -. forming the potent peroxynitrite (ONOO-), which causes oxidative damage, nitration and S-nitrosylation of biomolecules. Furthermore, ONOO- oxidizes the NOS cofactor 5,6,7,8-tetrahydrobiopterin with the consequence of uncoupling NOS from NO synthesis thereby leading NOS to a superoxide producing proarteriosclerotic enzyme ( Förstermann, 2006 ). (VI) Exogenous NO released from DETA/NONOate causes overexpression of TGF-beta and extracellular matrix in cultured human coronary smooth muscle cells (A. Schmidt et al., 2003).

2.3. eNOS-independent sources of NO

The generation of NO is not restricted to NO-synthases. An endothelium-independent source of bioactive NO is the ingestion of dietary (inorganic) nitrate. Naturally occurring dietary nitrate (celery, cress, chervil, beetroot, spinach, rucula contain up to 250 mg NO/100 g fresh weight) elevate the tissue und blood plasma level of nitrite via bioconversion in the entero-salivary circulation. When nitrite is acidified, it yields HNO2, which decomposes to NO and other nitrogen oxides

NO 2 +  H + HNO 2 E1
2 HNO 2     N 2 O 2 +  H 2 O E2
N 2 O 2   NO  +  NO 2 E3

Studies have indicated that acid-catalysed nitrite reduction to NO can also take place in blood vessels and tissues already at a moderately low pH and within nitrite concentrations normally present in vivo.

The NO generated by eNOS has a half-life (T1/2) of 1-2 milliseconds and rapidly oxidizes to nitrate (NO2 -). Nitrate however is not a final end product of NO metabolism but can be a substrate for NOS-independent regeneration to NO (Benjamin et al., 1994; Lundberg et al., 1994). Therefore other sources of nitrate in mammalians can contribute to the formation of NO such as nitrate generated from commensal bacteria in the digestive tract or nitrate present in foodstuff. Thus, in a study of Milkowski (Milkowski et al., 2010) it was shown that the consumption of nitrite- and nitrate-rich food such as fruits, leafy vegetables, and cured meals along with antioxidants can compensate for any disturbance in endogenous NO. Regular intake of nitrate-containing food such as green leafy vegetables may ensure that blood and tissue levels of nitrite and NO pools are maintained at a level sufficient to compensate for any disturbances in endogenous NO synthesis. In several studies (Kapil et al., 2010a, 2010b; Tang et al., 2011) it was shown that nitrate supplementation or vegetable intake (such as beetroot juice) causes dose-dependent elevation in plasma nitrite concentration, elevation of cGMP concentration with a consequent decrease in blood pressure and reduction the risk of ischaemic stroke. The collective body of evidence suggests that food enriched with nitrate and nitrite provide significant health benefits with very little risk. The weak and inconclusive data on the cancer risk of nitrite/nitrate and processed meats are far outweighed by the health benefit of restoring NO homeostasis via dietary nitrite and nitrate (Tang et al., 2011).


3. Regulation of eNOS activity

3.1. Phosphorylation

eNOS synthesizes NO in a pulsatile Ca2+/calmodulin-dependent manner with eNOS activity markedly increasing when intracellular Ca2+ increases. Ca2+ induces the binding of calmodulin to the enzyme thus increasing the rate of electron transfer from NADPH to heme center (Hemmens & Mayer, 1998). However, eNOS can be activated by other stimuli as increased intracellular Ca2+. The best-established stimulus is the shear stress of flowing blood, which can increase enzyme activity. This activation is mediated by phosphorylation of the enzyme (Fig. 2). The eNOS protein can be phosphorylated on several Ser, Thr and Tyr residues. Two main changes in enzyme function have been found. Phosphorylation of Ser1177 stimulates the flux of electrons within the reductase domain and increases the Ca2+ sensitivity of the enzyme (Fleming & Busse, 2003). Several protein kinases participate in phosphorylation of eNOS at Ser1177. These kinases include Akt, protein kinase A, 5’-AMP activated protein kinase and calmodulin-dependent kinase II. A negative regulatory site for phosphorylation is Thr495 under non-stimulated conditions probably by protein kinase C. Thr495 interferes with the binding of calmodulin to the calmodulin-binding domain. Dephosphorylation of Thr495 is associated with stimuli such as histamine and bradykinine both elevating intracellular Ca2+ concentration. Dephosphorylation of Thr495 has also been shown to favour eNOS uncoupling (Lin et al., 2003). Other phosphorylation sites including Ser114, Ser633 and some Tyr residues are not known to have major consequences for enzyme activity (Fleming & Busse, 2003; Fleming, 2010).

eNOS-associated proteins such as caveolin, heat shock protein 90 or eNOS interacting proteins provide the scaffold for the formation of the eNOS protein complex and its intracellular location (Fleming & Busse, 2003).

eNOS levels in endothelial cells can be regulated by changes in eNOS mRNA stability.

3.2. Enhancers of NO availability

Statins. Statins are a group of compounds which lower LDL-cholesterol, are inhibiting the enzyme 3-hydroxy-3-methylglutaryl coenzyme A. Beside their lipid lowering property statins improve vascular relaxation, reduce vascular inflammation, reduce oxidative stress, decrease thrombosis and platelet aggregation ( E. Schulz et al., 2004 ; Sowers, 2003). These beneficial effects of statins are in part mediated by an effect on eNOS because they can be blocked by L-NMMA (L-NG-monomethylarginine), an inhibitor of eNOS (John, et al., 1998; Rosenson & Tangney, 1998). Statins increase the expression of eNOS via Rho isoprenylation (Laufs et al., 1998) or posttranslational mechanism (Kureishi et al., 2000).

Superoxide dismutase (SOD). Superoxide dismutase has a key antioxidant role by dismutation of O2 -. into oxygen and hydrogen peroxide. In humans, three forms of the enzyme are present (SOD1, SOD2 and SOD3). In the cardiovascular system, the action of extracellular SOD3 (Cu–Zn–SOD) lowers O2 -. and maintains vascular NO levels (Jung et al., 2007).

Figure 2.

Regulation of eNOS activity by intracellular Ca2+ and phosphorylation. An increase in intracellular Ca2+ (as produced by agonists such as histamine or bradykinin) leads to an enhanced binding of CaM (calmodulin) to the enzyme, which in turn displaces an auto-inhibitory loop and facilitates the flow of electrons from NADPH in the reductase domain to the heme in the oxygenase domain. There are several potential phosphorylation sites in eNOS, but most is known about the functional consequences of phosphorylation of Ser1177 (human eNOS sequence) in the reductase domain and Thr495 (human eNOS sequence) within the CaM-binding domain. In resting endothelial cells, Ser1177 is usually not phosphorylated. Phosphorylation is induced when the cells are exposed to fluid shear stress, estrogens, VEGF, insulin, or bradykinin. The kinases responsible for phosphorylation depend on the primary stimulus. Shear stress elicits the phosphorylation of Ser1177 by activating protein kinase A (PKA), estrogen and VEGF phosphorylate eNOS mainly via Akt, insulin probably activates both Akt and the AMP-activated protein kinase (AMPK), and the bradykinin-induced phosphorylation of Ser1177 is mediated by CaMKII. Phosphorylation of the Ser1177 residue increases the flux of electrons through the reductase domain and thus enzyme activity. The Thr495 residue of human eNOS tends to be constitutively phosphorylated in endothelial cells. Thr495 is a negative regulatory site, and its phosphorylation is associated with a decrease in enzyme activity. The constitutively active kinase that phosphorylates eNOS Thr495 is most probably protein kinase C (PKC). The phosphatase that dephosphorylates Thr495 appears to be protein phosphatase1. (Figure and legend from U. Förstermann, Pflügers Arch - Eur J Physiol, 2010;459:923-933).

Catalase. Catalase decomposes hydrogen peroxide to water and oxygen. Overexpression of catalase has protective effects in the cardiovascular system such as delayed development of arteriosclerosis (Yang et al., 2004) and inhibition of angiotensin II-induced aortic wall hypertrophy (Zhang et al., 2005).

Glutathion peroxidase (GPx). Several isoenzymes of GPx were found in mammals, the isoenzyme 1 being most abundant. In patients with coronary artery disease the activity of red blood cell GPx1 is inversely associated with the risk of cardiovascular events (Blankenberg et al., 2003). In ApoE-deficient mice, the deficiency of GPx1 leads to arteriosclerotic lesion progression (Torzewski et al., 2007).

Heme oxygenase (HO). In break down of heme CO, biliverdin and free ferrous iron are formed. The biliverdin is converted to bilirubin, which has radical-scavenging properties (Jiang et al., 2006). The carbon monoxide has antiproliferative and anti-inflammatory as well as vasodilatory properties (Morita, 2005).

Thioredoxin (Trx). Thioredoxin seems to exert most of its ROS-scavenging properties through Trx peroxidase (peroxiredoxin), which uses endogenous SH groups as reducing equivalents. Thioredoxin is present in endothelial- and vascular smooth muscle cells. It exerts its ROS-scavenging properties through Trx peroxidase. Trx scavens ROS and nitric peroxide, ONOO- (Yamawaki et al., 2003).

Paraoxonase (PON). The PON family of enzymes acts as vascular antioxidant defense and protects against coronary artery disease (Aviram et al., 1998). The PON1 and PON3 enzymes are synthesized in the liver and circulate in plasma associated with the high-density lipoprotein (HDL) fraction. The capacity of HDL in decreasing HDL and LDL lipid peroxidation largely depends on its PON1 content (Aviram et al., 1998). Deletion of the PON1 gene increases oxidative stress in mouse macrophages and aortae (Rozenberg et al., 2005). The enzyme has been shown to reduce ROS in human endothelial cells, vascular smooth muscle cells, and fibroblasts (Horke et al., 2007).


4. eNOS – A multiple cofactors-dependent enzyme

eNOS is a homodimer protein and consists of two subunits: (I) the alpha reductase domain which is able to transfer electrons from NADPH to FAD and FMN and can bind calmodulin for stimulation of electron transfer. It has a limited capacity to reduce molecular oxygen to superoxide (O2 -.) (Stuehr et al., 2001). (II) The oxygenase domain of eNOS is unable to bind the cofactor 5,6,7,8-tetrahydrobiopterin or L-arginine and can not catalyse NO production. The presence of heme allows for NOS dimerization and is the only cofactor that is essential for NOS for the interaction and coupling reductase and oxygenase domains. eNOS monomers are unable to bind the 5,6,7,8-tetrahydrobiopterin or the L-arginine and can not catalyse NO production. Under pathological conditions the molecular oxygen is no longer coupled to L-arginine reduction but results in the production of superoxide. This phenomenon is referred to eNOS uncoupling ( Förstermann & Münzel, 2006 ; Li et al., 2002b).


5. eNOS uncoupling

5.1. Molecular mechanisms leading to eNOS uncoupling

Various mechanisms can contribute to eNOS uncoupling (Fig. 3). Their inbalance causes eNOS dysfunction and cardiovascular risk. This has been shown by numerous clinical studies and for experimental animals. (I) Inhibition of eNOS activity. A lack or deficiency of eNOS disrupted at the calmodulin binding site resulted in enhanced arteriosclerosis or peripheral coronary arteriosclerosis and aortic aneurism in ApoE/eNOS double knock out mice (Chen et al., 2001; Hodgin et al., 2002; Knowles et al., 2000; Kuhlencordt et al., 2001). (II) eNOS uncoupling factors such as hypercholesterolemia, diabetes, smoking, hypertension are associated with endothelial dysfunction. Evidence for uncoupling of eNOS has been obtained in endothelial cells treated with LDL (Pritchard et al., 1995) in peroxynitrite-treated rat aorta (Laursen et al., 2001) and in spontaneously hypertensive rats (Li et al., 2006), in human diabetes (Heitzer et al., 2000) and streptozotocin-induced diabetic rats (Hink et al., 2001). (III) Arginine deficiency. L-arginine – the physiological substrate of eNOS – is a constituent amino acid and present in human blood plasma in a concentration of 113.6±14.6 µM (Psychogios et al., 2011). A decrease of L-arginine induced in hypercholesterolemia below physiological levels favours eNOS uncoupling and formation of ROS. Beside L-arginine the asymmetric dimethylated form of arginine (ADMA) is a major component of blood plasma in a concentration of 0.4-0.8 µM (Billecke et al., 2009) and acts as an endogenous inhibitor of eNOS.

Figure 3.

Scheme of an endothelial NO synthase (eNOS) whose oxygen reduction is uncoupled from NO synthesis. Oxidative stress is associated with endothelial dysfunction. ONOO− can oxidize BH4 to biologically inactive products such as trihydrobiopterin radical (BH3 ) or trihydrobiopterin radical cation protonated at N5 (BH3 H+). The BH3 radical can be converted to the quinonoid 6,7-[8H]-H2-biopterin (BH2), which also lacks biological activity. When ONOO− overwhelms the cell’s capacity to re-reduce these products to BH4, eNOS “uncouples” and reduces oxygen to O2− , but does not synthesize NO anymore. eNOS then contributes to oxidative stress in the cell. (Figure and legend from U. Förstermann, Pflügers Arch - Eur J Physiol, 2010;459:923-933).

ADMA is formed by dimethylation of protein-bound L-arginine and released by proteolysis. ADMA acts as a local competitor of L-arginine (Cooke, 2004; Maas, 2005). Arginase is an ubiquitous enzyme which catalyses the degradation of arginine to ornithine and urea. Two isoenzymes are found in mammals. Arginase I catalyses the final step of the urea cycle in liver. Arginase II is a mitochondrial enzyme that functions in L-arginine homeostasis and can be dysregulated by ox LDL (Ryoo et al., 2006) resulting in eNOS uncoupling. (IV) 5,6,7,8-tetrahydrobiopterin deficiency. 5,6,7,8-tetrahydrobiopterin deficiency causes eNOS dysfunction and uncoupling (Moens & Kass, 2006), if the primary function of 5,6,7,8-tetrahydrobiopterin such as both allosteric and redox function, the improvement, the binding affinity of L-arginine for eNOS and providing the second electron to the heme of eNOS are missing. These alterations have the consequence that the reduction of molecular oxygen still occurs at the heme site of eNOS but oxidation of the guanidine nitrogen of L-arginine is prevented so that the reduced oxygen is converted by the uncoupled eNOS to superoxide instead of NO and citrulline (Gao et al., 2007; Xia et al. 1998). Even the partially oxidized 5,6,7,8-tetrahydrobiopterin - the 7,8-tetrahydrobiopterin (BH2) - has no eNOS cofactor activity and is unable to prevent superoxide formation of eNOS (Gao et al., 2007). In addition, BH2 probably competes with BH4 for eNOS binding. Therefore the ratio BH4/BH2 is important for eNOS activity (Shinozaki et al., 1999; Vasquez-Vivar et al., 2002). Apparently a diminished BH4/BH2 level rather than BH4 deficiency is a molecular trigger for eNOS uncoupling (Crabtree et al., 2008). Normally the majority of BH4 is present in vascular endothelial cells (Antoniades et al., 2007; Katusic, 2001) in a concentration of 1.40 pM/106 cells. Intracellular BH4 concentration has been found under hypercholesterolemic conditions thus aortic BH4 levels are decreased by 50% in hypercholesterolemic ApoE knockout mice compared with wild-type mice (Ozaki et al., 2002), but also discrepant results are described (d'Uscio et al. 2003; d'Uscio & Katusic, 2006) apparently depending on the degree of hypercholesterolemia and differences in the level of oxidative stress. The tissue level of BH4 is determined by the balance of biosynthesis from GTP via de novo synthesis by GTP cyclo hydrolase (GCH-1) or by the salvage pathway from BH2 back to BH4 and degradation by oxidation of BH4 to BH2 (T.S. Schmidt & Alp, 2007) – a process that can be rapidly accelerated by peroxynitrite (Landmesser et al., 2003; Laursen et al., 2001; Zou et al. 2002).

The oxidase-mediated stress of BH4 can be increased by several ROS producing enzyme systems such as NADPH oxidase that plays a major role in vascular cells (Förstermann, 2008; Harrison et al., 2003; Schnabel & Blankenberg, 2007), by xanthine oxidase, cytochrome P450 monooxygenase and enzymes of the respiratory chain. Xanthine oxidase is generated from xanthine dehydrogenase by proteolysis. This enzyme is another potential source of ROS in vascular disease. The enzyme readily donates electrons to molecular oxygen, thereby producing O2 -. and hydrogen peroxide. Oxypurinol, an inhibitor of xanthine oxidase decreases O2 -. production and improves endothelium-dependent vascular relaxation to acetylcholine in blood vessels from hyperlipidemic animals (Ohara et al., 1993). This suggests a contribution of xanthine oxidase to endothelial dysfunction in early hypercholesterolemia. Experimental evidence suggests that endothelial cells themselves can express xanthine dehydrogenase (xanthine oxidase) and that this expression is regulated in a redox sensitive way depending on endothelial NADPH oxidase (McNally et al., 2003).

All these cited cofactors required for regulation eNOS activity depend on the physiological transcription and translation of the corresponding genes. These processes, however, are regulated by epigenetics. Epigentics refer to chromatin-based pathways including three distinct but highly interrelated mechanisms: DNA methylation, Histone density and posttranslational modifications. These factors together offer new perspectives on transcriptional control paradigm in vascular endothelial cells and provide a molecular basis for understanding how the environment impacts the genome to modified function and disease susceptibility (Yan et al., 2010).

5.2. Mechanisms leading to a loss of function of eNOS

Oxidative stress is associated with endothelial dysfunction. Mechanistically, superoxide derived from NADPH oxidases and/or xanthine oxidase may combine with NO formed by a still functional eNOS. This would lead to increased formation of peroxinitrite (Laursen et al., 2001). Peroxynitrite has been shown to oxidize BH4 to biological inactive products. Significant O2 -. production also occurs when concentrations of L-arginine fall below the levels required to saturate the enzyme. In these circumstances eNOS catalysis the uncoupled reduction to O2 leading to the production of O2 -. and/or H2O2. Whether L-arginine concentration ever becomes critical as a substrate in vivo appears questionable since the Km of eNOS for L-arginine is ~3 µM while the L-arginine plasma concentration is ~100 µM and a ~10-fold accumulation of L-arginine within cells (Closs et al., 2000).

5.3. eNOS uncoupling in arteriosclerosis

Under cardiovascular risk factors such as diabetes, hypertension, smoking, the enzymatic reduction of molecular oxygen by eNOS is no longer used for L-arginine conversion to citrulline and NO, but the uncoupling of oxidase and reductase chain of eNOS produced ROS via the NADPH domains. The cardiovascular risk factors initiate the eNOS uncoupling and this can occur before arteriosclerotic lesions can be detected. The eNOS uncoupling can be triggered by various mechanisms which include BH4 deficiency, shortage of L-arginine or HSP 90, inhibitory phosphorylation of eNOS on Thr495 (see above) eNOS redistribution to the cytosolic fraction of the cell, oxidation of the zinc-thiolate cluster in eNOS or elevated ADMA levels (Sud et al. 2008). Among all of these mechanisms the reaction BH4 to BH2 is probably a dominant factor, and BH4 deficiency seems to be the primary cause for eNOS uncoupling in pathophysiology. Some researchers have postulated that eNOS may exist in two separate pools: a coupled form and an uncoupled form. The coupled enzyme is associated with the membrane and is readily accessible to the "signalome" for activation and NO production, whereas the uncoupled enzyme may reside in the cytosol and produces superoxide (Gharavi et al., 2006; Sullivan et al., 2006). In eNOS overexpressing mice for example, there is clear evidence for eNOS uncoupling (i.e. eNOS-mediated ROS production). In the same mice, however, NO-generating activity is elevated 2-fold when compared with wild-type mice (the total eNOS protein levels are elevated 8-fold) (Bendall et al., 2005). Thus, it is possible that coupled eNOS and uncoupled eNOS may exist in the same tissue at the same time.

The principle mechanisms of vascular protection by eNOS-derived NO and the consequences of endothelial dysfunction and the concomitant eNOS uncoupling are listed in Tab. 2.

Table 2.

The vaso-protective effect of eNOS is not restricted to the control of arterial dilation and constriction. The NO released towards the vascular lumen is a potent inhibitor of platelet aggregation and adhesion. The expression and formation of the alpha and beta component of various integrins including the cell adhesion molecules ICAM and VCAM can also be inhibited and reduce the transendothelial migration of macrophages and T-lymphocytes known to be an early event in the development of arteriosclerosis and characteristic for its inflammatory phases. The uncoupled eNOS leads to an excessive production of superoxide (O2 -. ) and in turn to the formation of highly toxic peroxynitrite ( Förstermann & Münzel, 2006 ).


6. eNOS-independent production of reactive oxygen species in vascular disease

Beside the eNOS there are several enzymes that can produce ROS in the endothelial cells: NADPH oxidase, xanthine oxidase, and enzymes of the mitochondrial respiratory chain are of major importance.

NADPH oxidases. Several isoforms of ROS producing NADPH oxidase are present and active in the vascular wall. In arteriosclerotic arteries the NADPH oxidase subunits NOX 2 and NOX 4 (Sorescu et al., 2002) have been identified.

Xanthine oxidase (XO). Increased cholesterol levels have been shown to stimulate the release of xanthine oxidase from the liver into the circulation. The circulating xanthine oxidase than can associate with endothelial glycosaminoglycans (White et al., 1996) however endothelial cells themselves can express xanthine oxidase and the expression is regulated in a redox sensitive pathway depending on endothelial NADPH oxidase (McNally et al., 2003) (Fig. 4).

Respiratory chain of the mitochondria. The molecular oxygen is consumed by mitochondria thereby forming O2 -.. Evidence has been provided that some cardiovascular diseases are associated with mitochondrial dysfunction (Ramachandran et al., 2002) and the mitochondrial production of ROS may be linked to the development of early arteriosclerotic lesions.

Figure 4.

XO and NOS are capable of generating either NO or superoxide (O2) depending on the conditions. When the supply of L-arginine and oxygen is good, NOS makes NO, whereas the same enzyme may generate considerable amounts of superoxide when L-arginine or cofactors are limited. XO generates superoxide, for example, during reperfusion after ischemia, whereas nitrite reduction to NO occurs preferentially during hypoxia. NO generation from XO can be beneficial and works as a backup system to supply NO during hypoxia when NO synthesis from NOS is compromised. Detrimental effects of these 2 enzyme systems can also be foreseen, for example, in a situation in which NO and superoxide are generated simultaneously and react to form potentially harmful peroxynitrite. (Figure and legend from J.O. Lundberg and E. Weitzberg, Arterioscler Thromb Vasc Biol, 2005;25:915 – 922).


7. Factors protecting against eNOS uncoupling and oxidative stress

7.1. Nitric oxide donors

NO-delivering drugs (NO donors) are used for their potential therapeutic benefit in coronary heart disease risk patients (D.J. Lefer & A.M. Lefer, 1988) by increasing coronary blood flow and dilating coronary arteries. Several studies have described the action of NO donors on vascular smooth muscle cells (Sarkar et al., 1997; A. Schmidt et al. 2003; Young et al., 2000). The pathway leading to NO formation differs among individual NO donor classes: indirect NO donors such as organic nitrates (nitroglycerol, isosorbide mononitrate, isosorbide dinitrate) require enzymatic catalysis, other NO donors require interaction with thiols to release NO, some have to undergo oxidation or reduction. In contrast, direct NO donors generate NO non-enzymatically. Examples are nicorandil, SIN-1 (the active metabolite of molsidomine) and the group of 1-substituted diazen-1-ium-1,2-diolates that releases NO spontaneously with a half-life from minutes to hours (Mooradian et al., 1995).

7.2. The NO donor DETA/NONOate

The compound (Z)-1-[2-Aminoethyl)–N–(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate (in the following detNO) belongs to the class of direct NO donors. Under cell culture conditions detNO releases spontaneously NO with a half-life of about 20 h at 37 C in a strictly first order reaction (Hrabie et al., 1993; Keefer et al., 1996; Mooradian et al., 1995), thereby disintegrating to two NO and diethylentriamine. Diethylentriamine, the byproduct of detNO disintegration, is known to be effectiveless (Mooradian et al., 1995; Sarkar et al., 1997). detNO has been successful used (Boyle et al., 2002; Ishimaru et al., 2001; A. Schmidt et al., 2003). In experimental studies (A. Schmidt et al., 2008) on cultured endothelial cells exogenously applied NO released from the NO donor detNO has a dual function in the regulation of eNOS expression. During short–term exposure of endothelial cells, exogenous detNO enhances the phosphorylation of the protein kinase Akt that in turn activates eNOS of endothelial cells by increasing its phosphorylation leading to a higher release of endogenous NO.

Phosphorylation can be achieved by exposure of human vascular endothelial cells to 150 µmol/L detNO. In short-term experiments in Western blot analysis detNO shows a clear increase of eNOS phosphorylation at Ser1177 after a short lag phase, detectable 20 min after detNO addition. The phosphorylation is mediated by the protein kinase Akt that is converted into p-Akt within 10 min after addition of detNO in a concentration-dependent manner. The phosphorylated Akt increases in turn Ser1177 phosphorylation of eNOS. This phosphorylation cascade could be reverted by preincubation of the cells with the PI-3 kinase inhibitor LY294002 that prevents phosphorylation of both Akt and eNOS. Thr495 is constitutively phosphorylated in all endothelial cells (Fleming & Busse, 2003) and is a negative regulatory site, i.e. phosphorylation leads to a decrease of eNOS activity. The release of endogenous NO in response to exogenous detNO was confirmed by L-[2,3,4,5-3H]arginine as indicator. The eNOS-mediated conversion of [3H]arginine to NO and [3H]citrulline was measured and the results are given in [3H]citrulline equivalents. A statistically significant increase of endogenous NO production after 20 and 30 min exposure to detNO is shown. N-nitro-L-arginine methyl ester HCl (NAME), a competitive NOS inhibitor, verifies the reaction conditions of the assay. Taking this reaction sequence into account, the effect of the NO donor could be considered partially a trigger for the acceleration of endogenous NO production that finally effects vasodilation via the physiologic pathway. This leads to the hypothesis of a potential switch from an exogenously applied to an endogenously generated NO stimulation (Fig. 5).

7.3. Long-term application of detNO and other NO donors

In contrast an exposure of endothelial cells to detNO for 24 and 48 h reduces the eNOS protein content as compared with controls. Densitometry revealed a reduced eNOS protein content after 24 h and 48 h. Real-time RT-PCR confirmed the reduced transcription of eNOS-specific mRNA. For direct determination of the reduced eNOS enzyme activity after long-term exposure to detNO, [2,3,4,5-3H]arginine was added to the culture medium. The radioactivity of [3H]citrulline formed by the NADPH-dependent NOS oxidoreductase is direct proportional to the NO produced and released by the endothelial cells. Under these conditions the results show a significant reduction of NO production expressed as [3H]citrulline equivalents in accordance to the reduced Ser1177 phosphorylation of eNOS. Taken together, these results emphasize a limitation of NO donors as long-term therapeutics owing to the inhibition of eNOS synthesis. However, whether exogenous NO donors are operative and effective in a similar way also in humans is still uncertain. In numerous clinical studies the outcome of repeated administration of indirect or direct NO donors to patients with coronary artery disease were ambiguous and the potential benefit of long-acting nitrates has remained controversial. Pathways leading to NO formation differ significantly among individual NO donor classes.

Figure 5.

Scheme of the new mechanism of action of the NO-donor DETA/NONOate in cultured human vascular endothelial cells. During short-term exposure the exogenous NO donor enhances the phosphorylation of the protein kinase Akt (PKB) that in turn activates eNOS of the endothelial cells by increasing its phosphorylation of Ser1177 leading to a higher release of the physiological endogenous NO as judged by the conversion of [3H]arginine to [3H]citrulline. The NOS-released NO reaches the smooth muscle cell and effects vasodilation.

In the Fourth International Study of Infarct Survival (ISIS-4), there was no significant reduction in five-week mortality and no survival advantage (ISIS-4 (Fourth International Study of Infarct Survival) Collaborative Group, 1995). Chronic administration of long acting nitrates in patients with healed myocardial infarction resulted in an increased number of patients with cardiac events (Ishikawa et al., 1996) and an increased risk of cardiac deaths occurred in CAD patients with long acting nitrates (Nakamura et al., 1999). Furthermore, a study on 19 healthy volunteers documented that isosorbide mononitrate given over 7 days impaired endothelial function due to formation of free radicals (Thomas et al., 2007). In total, epidemiological evidence indicates that chronic administration of long acting nitrates increase rather than decreases fatal and non-fatal events (Ishikawa et al., 1996; Nakamura et al., 1999). This view is confirmed by experiments on human vascular endothelial cells, which show detNO-induced cell cycle arrest and hypertrophy. Cultured quiescent EC released from the Go-phase by seeding at a low density re-enter the cell cycle and proliferate up to confluence. In this phase detNO causes a dose-dependent suppression of proliferation of EC indicated by a decreased incorporation of [3H]thymidine and a cell cycle arrest. The antiproliferative effect of detNO was associated with a remarkable increase of cell protein content that continued up to a 2-3-fold amount of control cells within 3 days while the cell number indicates an inhibition of cell proliferation and shows neither increase nor decrease. The elevated total cell protein was the result of de novo synthesis indicated by measurements of [3H]leucine incorporation into total cellular protein. After 48 h incubation of subconfluent cultures in the presence of [4,5-3H]leucine the incorporated radioactivity was 24.8 x 103 dpm/105 control cells and 34.6 x 103 dpm/105 cells exposed to detNO. The concomitant upregulation of p21 refers to a block at the G1-phase of the cell cycle. The detNO-induced metabolic alterations convert the cells into a hypertrophic phenotype. Measurements of cell volume show an increase from 2.49 ± 0.18 up to 3.38 ± 0.36 (200 µmol/L detNO) fL /cell.

The inhibition of proliferation is cytostatic but not cytotoxic as evaluated by cell death determination and is reversible. A quantitative determination of mono- and oligonucleosomes revealed no significant apoptotic cell death in detNO-pretreated cells. When the medium of detNO-induced growth-arrested cells is replaced by a standard medium, cell proliferation recovers within the following 48 h with continuous increase of cell number.

7.4. Antioxidant compounds potentially protecting against vascular oxidative stress

Important antioxidant enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, heme oxygenase (HO), and the thioredoxin (Trx) peroxidase and perhaps also paraoxonases (PON) (see Chapter 3.2).

Pentaerythritol tetranitrate (PETN) is a NO donor that does not induce significant nitrate tolerance and reduces oxidative stress (probably by inducing heme oxygenase). Sepiapterin can be reduced in cells by sepiapterin reductase (SR) to 7,8-BH2, sepiapterin reductase catalyses the following reaction

7 , 8 dihydrobiopterin  +  NADP +       tetrahydrobiopterin  +  NADPH  +  H + E4

Midostaurin, betulinic acid and ursolic acid upregulate eNOS and concomitantly decrease NADPH oxidase expression (Li & Förstermann, 2009).

AVE9488 and AVE3085, a new class of eNOS enhancers, upregulated the promotor activity of eNOS in vitro and in vivo. Application to wild type and ApoE-knockout mice over 12 weeks enhanced vascular eNOS expression at mRNA and protein level (Xue et al., 2010). The hybrid NO-releasing prodrug PABA/NO can be stabilized as nanoparticle with significant stability in mice circulation over 24h (Kumar et al., 2010).

Nebivolol - a NO-releasing beta-blocker induced a consistent increase of aortic eNOS expression rabbits receiving high-cholesterol diet (de Nigris et al., 2008). NO-releasing S-nitrosothiol-modified xerogels are capable of generating NO for up to 2 weeks (Riccio et al., 2009). These new generation of NO donors might be a rational approach to develop a new generation of antiatherogenic and anti-inflammatory NO donors. AVE9488 and AVE3085 are eNOS transcription enhancers that reverse eNOS uncoupling and preserve eNOS functionality.

Angiotensin II type 1-receptor blockers (ARBs), estrogens and erythropoietin (EPO) enhance (6R)-5,6,7,8-BH4 synthesis by stimulating GCH1 expression or activity (Li & Förstermann, 2009.)

Angiotensin-converting enzyme (ACE) inhibitors, the aldosterone antagonist eplerenone and the renin inhibitor aliskiren prevent (6R)-5,6,7,8-BH4 oxidation by decreasing the expression and/or activity of NADPH oxidase.


8. Clinical implications

Cardiovascular risk factors cause oxidative stress that alters the endothelial cells capacity and leads to endothelial dysfunction. The term „endothelial dysfunction“ is used to refer to an incompetence of endothelial cell-dependent vasorelaxation resulting from eNOS uncoupling but a molecular or biochemical basis for biomarkers indicating uncoupled eNOS has not been established. A biomarker is a characteristic that is objectively measured and evaluated as an indicator for normal or pathogenic processes or pharmacological response to a therapeutic intervention. As biomarkers for cardiovascular diseases oxLDL, CRP, IL-6, fibrinogen, TNF-alpha, MMP-9, MPO and cell adhesion molecules have been proposed (Vasan, 2006). Indirect biomarkers for eNOS uncoupling are a number of pharmaceuticals that have been shown to act as vaso-protective agents. Such agents listed by Förstermann (Förstermann, 2010) are: pentaerythritol tetranitrate, a NO donor that does not induce significant nitrate tolerance and reduces oxidative stress probably by inducing heme oxygenase 1, L-arginine stimulates NO release from eNOS, folic acid may improve eNOS functionality by stabilising BH4 and stimulating the endogenous regeneration of BH2 back to BH4, sepiapterin can be reduced in cells by sepiapterin reductase to BH2 and further dihydrofolate reductase to form BH4, midostaurin, betulinic acid and ursolic acid upregulate eNOS and concomitantly decrease NADPH oxidase expression, AVE9488 and AVE3085 are eNOS transcription enhancers that reverse eNOS uncoupling and preserve eNOS functionality, statins, angiotensin II type 1-receptor blockers, estrogens and erythropoietin enhance BH4 synthesis by stimulating GTP cyclohydrolase1 expression or activities. Statins, angiotensin converting enzyme inhibitors, the aldosterone antagonist eplerenone and the renin inhibitor prevent BH4 oxidation by decreasing the expression and/or activity of NADPH oxidase. All these compounds are secondary biomarkers indicating a pharmacological response to a therapeutic intervention.

Clinically, endothelial function can be assessed by invasive or non-invasive techniques (for review see Esper et al., 2006). These techniques evaluate the endothelial functional capacity depending on the amount of NO produced and the resulting vasodilation effect. The percentage of vasodilation with respect to the basal value represents the endothelial functional capacity. A non-invasive technique most often used is the transient flow-modulate endothelium-dependent post-ischemic vasodilation performed on conductance arteries such as the brachial, radial or femoral arteries. This vasodilation is compared with the vasodilation produced by NO donors. The vasodilation is quantified by measuring the arterial diameter with high-resolution ultrasonography. Laser-Doppler techniques are used to consider tissue perfusion. There is no doubt that endothelial dysfunction contributes to the initiation and progression of arteriosclerosis and could be considered an independent vascular risk factor.


9. Conclusion

Nitric oxide produced in vascular endothelial cells by the nitric oxide synthase is a major signalling molecule for maintaining vascular homeostasis. The nitric oxide synthase - constitutionally expressed by endothelial cells – is a dimeric enzyme molecule depending on multiple cofactors for its physiological activity and optimal endothelial function. Any imbalance of reductase and NADPH oxygenase or deficient supply of the enzyme substrate L-arginine or of cofactors leads to an upregulation of endothelial nitric oxide synthase and oxygenase activity with the consequence of an uncoupling of the nitric oxide synthase and production of detrimental reactive oxygen species and/or highly toxic peroxinitrate instead of nitric oxide. The resulting endothelial dysfunction implies a high cardiovascular risk. Several drugs reverting endothelial nitric oxide synthase uncoupling and/or improving endothelial dysfunction are in clinical use. Nitric oxide delivering drugs (NO donors) show potential therapeutical benefit and are used to relief or prevent acute episodes of angina pectoris by activating the endothelial nitric oxide synthase – a new mechanism found for the NO donor DETA/NONOate. However, a long-term administration of NO donors has been found to reduce endothelial nitric oxide synthase of endothelial cells drastically (in cell culture experiments). This could be the basis for development of a new generation of NO donors that mimics the low continuous pulsatile stress-induced release of endogenous nitric oxide.



The author wishes to thank Prof. Dr. E. Buddecke, Muenster, for critical discussion and revising the manuscript.


  1. 1. Antoniades C. Shirodaria C. Crabtree M. Rinze R. Alp N. Cunnington C. Diesch J. Tousoulis D. Stefanadis C. Leeson P. Ratnatunga C. Pillai R. Channon K. M. 2007 Altered plasma versus vascular biopterins in human atherosclerosis reveal relationships between endothelial nitric oxide synthase coupling, endothelial function, and inflammation. Circulation, 116 24 2851 2859
  2. 2. Aviram M. Rosenblat M. Bisgaier C. L. Newton R. S. Primo-Parmo S. L. La Du B. N. 1998 Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase. J Clin Invest, 101 1581 1590
  3. 3. Bendall J. K. Alp N. J. Warrick N. Cai S. Adlam D. Rockett K. Yokoyama M. Kawashima S. Channon K. M. 2005 Stoichiometric relationships between endothelial tetrahydrobiopterin, endothelial NO synthase (eNOS) activity, and eNOS coupling in vivo: insights from transgenic mice with endothelialtargeted GTP cyclohydrolase 1 and eNOS overexpression. Circ Res, 97 9 864 871
  4. 4. Benjamin N. O’Driscoll F. Dougall H. Duncan C. Smith L. Golden M. McKenzie H. 1994 Stomach NO synthesis. Nature, 368:502
  5. 5. Billecke S. S. D’Alecy L. G. Platel R. Whitesall S. E. Jamerson K. A. Perlman L. Gadegbeku C. A. 2009 Blood content of asymmetric dimethylarginine: new insights into its dysregulation in renal disease. Nephrol Dial Transplant, 24 2 489 496
  6. 6. Blankenberg S. Rupprecht H. J. Bickel C. Torzewski M. Hafner G. Tiret L. Smieja M. Cambien F. Meyer J. Lackner K. J. AtheroGene Investigators 2003 Glutathione peroxidase 1 activity and cardiovascular events in patients with coronary artery disease. N Engl J Med, 349 17 1605 1613
  7. 7. Boyle J. J. Weissberg P. L. Bennett M. R. 2002 Human macrophage- induced vascular smooth muscle cell apoptosis requires NO enhancement of Fas/Fas-L interactions. Arterioscler Thromb Vasc Biol, 22 1624 1630
  8. 8. Busse R. Luckhoff A. Bassenge E. 1987 Endotheliumderived relaxant factor inhibits platelet activation. Naunyn-Schmiedeberg’s Arch Pharmacol, 336 566 571
  9. 9. Castel H. Vaudry H. 2001 Nitric oxide directly activates GABA (A) receptor function through a cGMP/protein kinase-independent pathway in frog pituitary melanotrophs. J Neuroendocrinol, 13 695 705
  10. 10. Chen J. Kuhlencordt P. J. Astern J. Gyurko R. Huang P. L. 2001 Hypertension does not account for the accelerated atherosclerosis and development of aneurysms in male apolipoprotein e/endothelial nitric oxide synthase double knockout mice. Circulation, 04 20 2391 2394
  11. 11. Clancy R. M. Leszczynska P. Piziak J. Abramson S. B. 1992 Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on NADPH oxidase. J Clin Invest, 90 1116 1121
  12. 12. Closs E. I. Scheld J. S. Sharafi M. Förstermann U. 2000 Substrate supply for nitric- oxide synthase in macrophages and endothelial ells: role of cationic amino acid transporters. Mol Pharmacol, 57 68 74
  13. 13. Cooke J. P. 2004 Asymmetrical dimethylarginine: the Uber marker? Circulation, 109 15 1813 1818
  14. 14. Crabtree M. J. Smith C. L. Lam G. Goligorsky M. S. Gross S. S. 2008 Ratio of 5,6,7,8- tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS. Am J Physiol Heart Circ Physiol, 294:H1530-1540
  15. 15. de Nigris F. Mancini F. P. Balestrieri M. L. Byrns R. Fiorito C. Wiiliams-Ignarro S. Palagiano A. Crimi E. Ignarro L. J. Napoli C. 2008 Therapeutic dose of nebivolol, a nitric oxide-releasing beta-blocker, reduces atherosclerosis in cholesterol-fed rabbits. Nitric Oxide, 19 1 57 63
  16. 16. d’Uscio L. V. Milstien S. Richardson D. Smith L. Katusic Z. S. 2003 Longterm vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res, 92 88 95
  17. 17. d’Uscio L. V. Katusic Z. S. 2006 Increased vascular biosynthesis of tetrahydrobiopterin in apolipoprotein E-deficient mice. Am J Physiol Heart Circ Physiol, 290:H2466 2471
  18. 18. Esper R. J. Nordaby R. A. Vilarino J. O. Paragano A. Cacharrón J. L. Machado R. A. 2006 Endothelial dysfunction: a comprehensive appraisal. Cardiovasc Diabetol, 5:4 doi:10.1186/1475-2840-5-4
  19. 19. Fleming I. Busse R. 2003 Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol, 284:R1 12
  20. 20. Fleming I. 2010 Molecular mechanisms underlying the activation of eNOS. Pflügers Arch-Eur J Physiol, 459 793 806
  21. 21. Förstermann U. Closs E. I. Pollock J. S. Nakane M. Schwarz P. Gath I. Kleinert H. 1994 Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension,.23(6, Part 2):1121-1131
  22. 22. Förstermann U. Münzel T. 2006 Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation, 113 1708 1714
  23. 23. Förstermann U. 2006 Janus-faced role of endothelial NO synthase in vascular disease: uncoupling of oxygen reduction from NO synthesis and its pharmacological reversal. Biol Chem, 387 1521 1533
  24. 24. Förstermann U. 2008 Oxidative stress in vascular disease: causes, defense mechanisms and potential therapies. Nat Clin Pract Cardiovasc Med, 5 338 349
  25. 25. Förstermann U. 2010 Nitric oxide and oxidative stress in vascular disease Pflügers Arch-Eur J Physiol, 459 923 939
  26. 26. Gao Y. T. Roman L. J. Martasek P. Panda S. P. Ishimura Y. Masters B. S. 2007 Oxygen metabolism by endothelial nitric-oxide synthase. J Biol Chem, 282 28565 28557 28565
  27. 27. Gharavi N. M. Baker N. A. Mouillesseaux K. P. Yeung W. Honda H. M. Hsieh X. Yeh M. Smart E. J. Berliner J. A. 2006 Role of endothelial nitric oxide synthase in the regulation of SREBP activation by oxidized phospholipids. Circ Res, 98 6 768 776
  28. 28. Harrison D. Griendling K. K. Landmesser U. Hornig B. Drexler H. 2003 Role of oxidative stress in atherosclerosis. Am J Cardiol, 91:7A 11A
  29. 29. Heitzer T. Krohn K. Albers S. Meinertz T. 2000 Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with Type II diabetes mellitus. Diabetologia, 43 1435 1438
  30. 30. Hemmens B. Mayer B. 1998 Enzymology of nitric oxide synthases. Methods Mol Biol, 100 1 32
  31. 31. Herman A. G. Moncada S. 2005 Therapeutic potential of nitric oxide donors in the prevention and treatment of atherosclerosis. Eur Heart J, 26 19 1945 1955
  32. 32. Hink U. Li H. Mollnau H. Oelze M. Matheis E. Hartmann M. Skatchkov M. Thaiss F. Stahl R. A. K. Warnholtz A. Meinertz T. Griendling K. Harrison D. G. Förstermann U. Münzel T. 2001 Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res, 88(2):E14 E22
  33. 33. Hodgin J. B. Knowles J. W. Kim H. S. Smithies O. Maeda N. 2002 Interactions between endothelial nitric oxide synthase and sex hormones in vascular protection in mice. J Clin Invest, 109 541 548
  34. 34. Horke S. Witte I. Wilgenbus P. Kruger M. Strand D. Förstermann U. 2007 Paraoxonase-2 reduces oxidative stress in vascular cells and decreases endoplasmic reticulum stress-induced caspase activation. Circulation, 115 2055 2064
  35. 35. Hrabie J. A. Klose J. R. Wink D. A. Keefer L. K. 1993 New nitric oxide- releasing zwitterions derived from polyamines. J Org Chem, 58 1472 1476
  36. 36. Ishikawa K. Kanamasa K. Ogawa I. Takenaka T. Naito T. Kamata N. Yamamoto T. Nakai S. Hama J. Oyaizu M. Kimura A. Yamamoto K. Aso N. Arai M. Yabushita H. Katori Y. 1996 Long-term nitrate treatment increases cardiac events in patients with healed myocardial infarction. Secondary Prevention Group. Jpn Circ J, 60 10 779 788
  37. 37. Ishimaru R. S. Leung K. Hong L. La Polt P. S. 2001 Inhibitory effects of nitric oxide on estrogen production and cAMP levels in rat granulose cell cultures. J Endocrinol, 168 249 255 ISIS-4 (Fourth International Study of Infarct Survival) Collaborative Group. (1995). ISIS-4: arandomised factorial trial assessing early oral captopril, oral mononitrate, and intravenous magnesium sulphate in 58,050 patients with suspected acute myocardial infarction. Lancet, 345:669-685
  38. 38. Jiang F. Roberts S. J. Datla S. Dusting G. J. 2006 NO modulates NADPH oxidasefunction via heme oxygenase-1 in human endothelial cells. Hypertension 48 950 957
  39. 39. John S. Schlaich M. Langenfeld M. Weihprecht H. Schmitz G. Weidinger G. Schmieder R. E. 1998 Increased bioavailability of nitric oxide after lipidlowering therapy in hypercholesterolemic patients: a randomized, placebo-controlled, double-blind study. Circulation,211 216
  40. 40. Jung O. Marklund S. L. Xia N. Busse R. Brandes R. P. 2007 Inactivation of extracellular superoxide dismutase contributes to the development of high-volume hypertension. Arterioscler Thromb Vasc Biol, 27 470 477
  41. 41. Kapil V. Milsom A. B. Okorie M. Maleki-Toyserkani S. Akram F. Rehman F. Arghandawi S. Pearl V. Benjamin N. Loukogeorgakis S. Macallister R. Hobbs A. J. Webb A. J. Ahluwalia A. 2010a Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO. Hypertension, 56 2 274 281
  42. 42. Kapil V. Webb A. J. Ahluwalia A. 2010b Inorganic nitrate and the cardiovascularsystem. Heart, 96 21 1703 1709
  43. 43. Katusic Z. S. 2001 Vascular endothelial dysfunction: does tetrahydrobiopterin play a role? Am J Physiol Heart Circ Physiol, 281: H981 986
  44. 44. Keefer L. K. Nims R. W. Davies K. M. Wink D. A. 1996 NONOates’ (1-substituteddiazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms. Meth Enzymol, 268 281 293
  45. 45. Knowles J. W. Reddick R. L. Jennette J. C. Shesely E. G. Smithies O. Maeda N. 2000 Enhanced atherosclerosis and kidney dysfunction in eNOS(-/-)Apoe(-/-) mice are ameliorated by enalapril treatment. J Clin Invest, 105 451 845
  46. 46. Kockx M. M. de Meyer G. R. Muhring J. Bult H. Bultinck J. Herman A. G. 1996 Distribution of cell replication and apoptosis in atherosclerotic plaques of cholesterol-fed rabbits. Atherosclerosis, 120 115 124
  47. 47. Kockx M. M. 1998 Apoptosis in the atherosclerotic plaque: quantitative and qualitativeaspects. Arterioscler Thromb Vasc Biol, 18 1519 1522
  48. 48. Kuhlencordt P. J. Gyurko R. Han F. Scherrer-Crosbie M. Aretz T. H. Hajjar R. Picard M. H. Huang P. L. 2001 Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice. Circulation, 104 4 448 454
  49. 49. Kumar V. Hong S. Y. Maciag A. E. Saavedra J. E. Adamson D. H. Prud’ homme R. K. Keefer L. K. Chakrapani H. 2010 Stabilization of the nitric oxide (NO) prodrugs and anticancer leads, PABA⁄NO and double JS-K, through incorporation into PEG-protected nanoparticles. Mol Pharm, 7 1 291 298
  50. 50. Kureishi Y. Luo Z. Shiojima I. Bialik A. Fulton D. Lefer D. J. Sessa W. C. Walsh K. 2000 The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med, 6 1004 1010
  51. 51. Landmesser U. Dikalov S. Price S. R. McCann L. Fukai T. Holland S. M. Mitch W. E. Harrison D. G. 2003 Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest, 111 8 1201 1209
  52. 52. Laufs U. La Fata V. Plutzky J. Liao J. K. 1998 Upregulation of endothelial nitric oxidesynthase by HMG CoA reductase inhibitors. Circulation, 97 1129 1135
  53. 53. Laursen J. B. Somers M. Kurz S. McCann L. Warnholtz A. Freeman B. A. Tarpey M. Fukai T. Harrison D. G. 2001 Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation, 103 9 1282 1288
  54. 54. Lefer D. J. Lefer A. M. 1988 Studies on the mechanism of the vasodilator action ofnicorandil. Life Sci, 42 1907 1914
  55. 55. Li H. Förstermann U. 2000 Nitric oxide in the pathogenesis of vascular disease. JPathol, 190 244 254
  56. 56. Li H. Wallerath T. Förstermann U. 2002a Physiological mechanisms regulating theexpression of endothelial-type NO synthase. Nitric Oxide, 7 132 147
  57. 57. Li H. Wallerath T. Münzel T. Förstermann U. 2002b Regulation of endothelial- typeNO synthase expression in pathophysiology and in response to drugs. Nitric Oxide, 7 149 164
  58. 58. Li H. Witte K. August M. Brausch I. Godtel-Armbrust U. Habermeier A. Closs El. Oelze M. Münzel T. Förstermann U. 2006 Reversal of endothelial nitric oxide synthase uncoupling and up-regulation of endothelial nitric oxide synthase expression lowers blood pressure in hypertensive rats. J Am Coll Cardiol, 47 12 2536 2544
  59. 59. Li H. Förstermann U. 2009 Prevention of Atherosclerosis by Interference with theVascular Nitric Oxide System. Curr Pharm Des, 15 27 3133 3145
  60. 60. Lin M. I. Fulton D. Babbitt R. Fleming I. Busse R. Pritchard K. A. Jr. Sessa W. C. 2003 Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem, 278 44719 44726
  61. 61. Lloyd-Jones I. M. Block K. D. 1996 The vascular biology of nitric oxide and its role inatherogenesis. Annu Rev Med,.47 365 375
  62. 62. Liu V. W. Huang P. L. 2008 Cardiovascular roles of nitric oxide: a review of insightsfrom nitric oxide synthase gene disrupted mice. Cardiovasc Res,.77 1 19 29
  63. 63. Lundberg J. O. Weitzberg E. Lundberg J. M. Alving K. 1994 Intragastric nitric oxideproduction in humans: measurements in expelled air. Gut. 35 1543 1546
  64. 64. Lundberg J. O. Weitzberg E. 2005 NO Generation From Nitrite and Its Role in VascularControl. Arterioscler Thromb Vasc Biol, 25 5 915 922
  65. 65. Maas R. 2005 Pharmacotherapies and their influence on asymmetric dimethylargine(ADMA). Vasc Med, 10(Suppl 1): S49 57
  66. 66. Mallis R. J. Buss J. E. Thomas J. A. 2001 Oxidative modification of H-ras: S-thiolationand S-nitrosylation of reactive cysteines. Biochem J, 355 145 153
  67. 67. McNally J. S. Davis M. E. Giddens D. P. Saha A. Hwang J. Dikalov S. Jo H. Harrison D. G. 2003 Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. Am J Physiol Heart Circ Physiol, 285:H2290 H2297
  68. 68. Milkowski A. Garg H. K. Coughlin J. R. Bryan N. S. 2010 Nutritional epidemiology inthe context of nitric oxide biology: A risk-benefit evaluation for dietary nitrite and nitrate. Nitric Oxide, 22 2 110 119
  69. 69. Moens A. L. Kass D. A. 2006 Tetrahydrobiopterin and cardiovascular disease. Arterioscler Thromb Vasc Biol, 26 2439 2444
  70. 70. Moncada S. Palmer R. M. Higgs E. A. 1991 Nitric oxide: physiology, pathophysiology,and pharmacology. Pharmacol Rev, 43 2 109 142
  71. 71. Mooradian D. L. Hutsell T. C. Keefer L. K. 1995 Nitric oxide (NO) donor molecules:effect of NO release rate on vascular smooth muscle cell proliferation in vitro. J Cardiovasc Pharmacol, 25 674 678
  72. 72. Morita T. 2005 Heme oxygenase and atherosclerosis. Arterioscler Thromb Vasc Biol, 25 1786 1795
  73. 73. Nakamura Y. Moss A. J. Brown M. W. Kinoshita M. Kawai C. 1999 Longterm nitrateuse may be deleterious in ischemic heart disease: A study using the databases from two large-scale postinfarction studies. Multicenter Myocardial Ischemia Research Group. Am Heart J, 138 577 585
  74. 74. Ohara Y. Peterson T. E. Harrison D. G. 1993 Hypercholesterolemia increasesendothelial superoxide anion production. J Clin Invest, 91 2546 2551
  75. 75. Ozaki M. Kawashima S. Yamashita T. Hirase T. Namiki M. Inoue N. Hirata K. Yasui H. Sakurai H. Yoshida Y. Masada M. Yokoyama M. 2002 Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J Clin Invest, 110 3 331 340
  76. 76. Pritchard K. A. Jr. Groszek L. Smalley D. M. Sessa W. C. Wu M. Villalon P. Wolin M. S. Stemerman M. B. 1995 Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res, 77 3 510 518
  77. 77. Psychogios N. Hau D. D. Peng J. Guo A. C. Mandal R. Bouatra S. Sinelnikov I. Krishnamurthy R. Eisner R. Gautam B. Young N. Xia J. Knox C. Dong E. Huang P. Hollander Z. Pedersen T. L. Smith S. R. Bamforth F. Greiner R. McManus B. Newman J. W. Goodfriend T. Wishart D. S. 2011 The human serum metabolome. PLoS One,16;6(2):e16957
  78. 78. Radomski M. W. Palmer R. M. Moncada S. 1987 The anti- aggregating properties ofvascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmacol, 92 639 646
  79. 79. Ramachandran A. Levonen A. L. Brookes P. S. Ceaser E. Shiva S. Barone M. C. Darley-Usmar V. 2002 Mitochondria, nitric oxide, and cardiovascular dysfunction. Free Radic Biol Med, 33 1465 1474
  80. 80. Riccio D. A. Dobmeier K. P. Hetrick E. M. Privett B. J. Paul H. S. Schoenfisch M. H. 2009 Nitric oxide-releasing S-nitrosothiol-modified xerogels. Biomaterials, 30 4494 502
  81. 81. Rosenson R. S. Tangney C. C. 1998 Antiatherothrombotic properties of statins:implications for cardiovascular event reduction. JAMA, 279 1643 1650
  82. 82. Rozenberg O. Shih D. M. Aviram M. 2005 Paraoxonase 1 (PON1) attenuatesmacrophage oxidative status: studies in PON1 transfected cells and in PON1 transgenic mice. Atherosclerosis, 181 9 18
  83. 83. Ryoo S. Lemmon C. A. Soucy K. G. Gupta G. White A. R. Nyhan D. Shoukas A. Romer L. H. Berkowitz D. E. 2006 Oxidized low-density lipoprotein-dependent endothelial arginase II activation contributes to impaired nitric oxide signaling. Circ Res, 99 9 951 960
  84. 84. Sarkar R. Gordon D. Stanley J. C. Webb R. C. 1997 Cell cycle effects of nitric oxide onvascular smooth muscle cells. Am J Physiol, 272:H1810 1818
  85. 85. Schmidt A. Geigenmüller S. Völker W. Seiler P. Buddecke E. 2003 Exogenousnitric oxide causes overexpression of TGF-beta1 and overproduction of extracellular matrix in human coronary smooth muscle cells. Cardiovasc Res, 58 671 678
  86. 86. Schmidt A. Bilgasem S. Lorkowski S. Vischer P. Völker W. Breithardt G. Siegel G. Buddecke E. 2008 Exogenous nitric oxide regulates activity and synthesis of vascular endothelial nitric oxide synthase. Eur J Clin Invest, 38 7 476 485
  87. 87. Schmidt T. S. Alp N. J. 2007 Mechanisms for the role of tetrahydrobiopterin inendothelial function and vascular disease. Clin Sci (Lond), 113 47 63
  88. 88. Schnabel R. Blankenberg S. 2007 Oxidative stress in cardiovascular disease: successfultranslation from bench to bedside? Circulation, 116 1338 1340
  89. 89. Schulz E. Anter E. Keaney J. F. Jr. 2004 Oxidative stress, antioxidants, and endothelialfunction. Curr Med Chem, 11 1093 1104
  90. 90. Schulz R. Kelm M. Heusch G. 2004 Nitric oxide in myocardial ischemia/reperfusioninjury. Cardiovasc Res, 61 3 402 413
  91. 91. Shinozaki K. Kashiwagi A. Nishio Y. Okamura T. Yoshida Y. Masada M. Toda N. Kikkawa R. 1999 Abnormal biopterin metabolism is a major cause of impaired endothelium-dependent relaxation through nitric oxide/O2- imbalance in insulin-resistant rat aorta. Diabetes, 48 12 2437 2445
  92. 92. Sorescu D. Weiss D. Lassegue B. Clempus R. E. Szocs K. Sorescu G. P. Valppu L. Quinn M. T. Lambeth J. D. Vega J. D. Taylor W. R. Griendling K. K. 2002 Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation, 105 1429 1435
  93. 93. Sowers J. R. 2003 Effects of statins on the vasculature: implications for aggressive lipidmanagement in the cardiovascular metabolic syndrome. Am J Cardiol, 91:14B-22B
  94. 94. Stuehr D. Pou S. Rosen G. M. 2001 Oxygen reduction by nitric- oxide synthases. J BiolChem, 276 14533 14536
  95. 95. Sud N. Wells S. M. Sharma S. Wiseman D. A. Wilham J. Black S. M. 2008 Asymmetric dimethylarginine inhibits HSP90 activity in pulmonary arterial endothelial cells: role of mitochondrial dysfunction. Am J Physiol Cell Physiol, 294:C1407 1418
  96. 96. Sullivan J. C. Pollock J. S. 2006 Coupled and uncoupled NOS: separate but equal?Uncoupled NOS in endothelial cells is a critical pathway for intracellular signaling. Circ Res, 98 717 719
  97. 97. Sun J. Xin C. Eu J. P. Stamler J. S. Meissner G. 2001 Cysteine-3635 is responsible forskeletal muscle ryanodine receptor modulation by NO. Proc Natl Acad Sci USA, 98 1158 1162
  98. 98. Tang Y. Jiang H. Bryan N. S. 2011 Nitrite and nitrate: cardiovascular risk-benefit andmetabolic effect. Curr Opin Lipidol, 22 1 11 15
  99. 99. Thomas G. R. Di Fabio J. M. Gori T. Parker J. D. 2007 Once daily Therapy WithIsosorbide-5-Mononitrate Causes Endothelial Dysfunction in Humans. J Am Coll Cardiol, 49 1289 1295
  100. 100. Torzewski M. Ochsenhirt V. Kleschyov A. L. Oelze M. Daiber A. Li H. Rossmann H. Tsimikas S. Reifenberg K. Cheng F. Lehr H. A. Blankenberg S. Förstermann U. Münzel T. Lackner K. J. 2007 Deficiency of glutathione peroxidase-1 accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol, 27 850 857
  101. 101. Vasan R. S. 2006 Biomarkers of Cardiovascular Disease: Molecular Basis and PracticalConsiderations. Circulation, 113 2335 23362
  102. 102. Vasquez-Vivar J. Duquaine D. Whitsett J. Kalyanaraman B. Rajagopalan S. 2002 Altered tetrahydrobiopterin metabolism in atherosclerosis: implications for use of oxidized tetrahydrobiopterin analogues and thiol antioxidants. Arterioscler Thromb Vasc Biol, 22 1655 1661
  103. 103. White C. R. Darley-Usmar V. Berrington W. R. McAdams M. Gore J. Z. Thompson J. A. Parks D. A. Tarpey M. M. Freeman B. A. 1996 Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits. Proc Natl Acad Sci USA, 93 8745 8749
  104. 104. Xia Y. Tsai A. L. Berka V. Zweier J. L. 1998 Superoxide Generation from endothelialnitric-oxide synthase. A a2+/calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol Chem, 273 25804 25808
  105. 105. Xue H. M. He G. W. Huang J. H. Yang Q. 2010 New strategy of endothelial protectionin cardiac surgery: use of enhancer of endothelial nitric oxide synthase. World J Surg, 34 1461 1469
  106. 106. Yamawaki H. Haendeler J. Berk B. C. 2003 Thioredoxin: a key regulator ofcardiovascular homeostasis. Circ Res, 93 1029 1033
  107. 107. Yan M. S. Matouk C. C. Marsden P. A. 2010 Epigenetics of the vascular endothelium. JAppl Physiol, 109 3 916 926
  108. 108. Yang H. Roberts L. J. Shi M. J. Zhou L. C. Ballard B. R. Richardson A. Guo Z. M. 2004 Retardation of atherosclerosis by overexpression of catalase or both Cu/Zn-superoxide dismutase and catalase in mice lacking apolipoprotein E. Circ Res, 95 1075 1081
  109. 109. Young D. V. Serebryanik D. Janero D. R. Tam S. W. 2000 Suppression of proliferationof human coronary artery smooth muscle cells by the nitric oxide donor, S-nitrosoglutathione, is cGMP-independent. Mol Cell Biol Res Commun, 4 32 36
  110. 110. Zhang Y. Griendling K. K. Dikalova A. Owens G. K. Taylor W. R. 2005 Vascularhypertrophy in angiotensin II-induced hypertension is mediated by vascular smooth muscle cell-derived H2O2. Hypertension. 46 732 737
  111. 111. Zou M. H. Shi C.. Cohen R. A. 2002 Oxidation of the zinc- thiolate complex anduncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest, 109 817 826

Written By

Annette Schmidt

Submitted: May 5th, 2011 Published: March 14th, 2012